COMPOUNDS, PHARMACEUTICAL COMPOSITIONS, AND METHODS OF THEIR USE IN REVERSING CANCER CHEMORESISTANCE
20220280528 · 2022-09-08
Inventors
- Mo MOTAMEDI (Boston, MA, US)
- Junichi HANAI (Boston, MA, US)
- Vikas SUKHATME (Boston, MA, US)
- Mohammad MOVASSAGHI (Cambridge, MA, US)
Cpc classification
A61P31/00
HUMAN NECESSITIES
A61K31/548
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K31/548
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
C07D519/00
CHEMISTRY; METALLURGY
C07D513/22
CHEMISTRY; METALLURGY
International classification
Abstract
Described herein are compounds, pharmaceutical compositions, and methods for use in reversing cancer chemoresistance.
Claims
1. A method of killing a chemoresistant cancer cell, the method comprising (i) contacting the chemoresistant cancer cell with an effective amount of a compound selected from the group consisting of chaetocin, a chaetocin analogue, a Suv39H1/Suv39H2 inhibitor, and pharmaceutically acceptable salts thereof, and (ii) contacting the cell with an anti-cancer agent or a pharmaceutically acceptable salt thereof; wherein the chemoresistant cancer cell is a chemoresistant mesenchymal cancer cell or a chemoresistant cancer cell expressing Suv39H1/Suv39H2; and wherein step (ii) kills the chemoresistant cancer cell.
2. The method of claim 1, wherein the chemoresistant cancer cell is a chemoresistant mesenchymal cancer cell.
3. The method of claim 1, wherein the chemoresistant cancer cell is a chemoresistant cancer cell expressing Suv39H1/Suv39H2.
4. A method of treating a chemoresistant cancer in a subject, the method comprising (i) administering to the subject in need thereof a therapeutically effective amount of a compound selected from the group consisting of chaetocin, a chaetocin analogue, a Suv39H1/Suv39H2 inhibitor, and pharmaceutically acceptable salts thereof, and, (ii) administering to the subject a therapeutically effective amount of an anti-cancer agent or a pharmaceutically acceptable salt thereof; wherein the chemoresistant cancer comprises a chemoresistant mesenchymal cancer cell or a chemoresistant cancer cell expressing Suv39H1/Suv39H2; and wherein step (ii) treats the chemoresistant cancer in the subject.
5. The method of claim 4, wherein the chemoresistant cancer comprises a chemoresistant mesenchymal cancer cell.
6. The method of claim 4, wherein the chemoresistant cancer comprises a chemoresistant cancer cell expressing Suv39H1/Suv39H2.
7. A method of killing a chemoresistant cancer cell, the method comprising (i) contacting the chemoresistant cancer cell with an effective amount of a compound selected from the group consisting of chaetocin, a chaetocin analogue, a Suv39H1/Suv39H2 inhibitor, and pharmaceutically acceptable salts thereof, and, (ii) after a period of at least 5 hours, contacting the cell with an anti-cancer agent or a pharmaceutically acceptable salt thereof; wherein step (ii) kills the chemoresistant cancer cell.
8. The method of claim 7, wherein the period is 5 hours to 2 weeks.
9. The method of claim 8, wherein the period is 5 hours to 1 week.
10. The method of claim 9, wherein the period is 5 hours to 6 days.
11. The method of claim 10, wherein the period is 5 hours to 2 days.
12. The method of claim 11, wherein the period is 5 to 15 hours.
13. The method of claim 7, wherein the period is at least 15 hours.
14. A method of treating a chemoresistant cancer in a subject, the method comprising (i) administering to the subject in need thereof a therapeutically effective amount of a compound selected from the group consisting of chaetocin, a chaetocin analogue, a Suv39H1/Suv39H2 inhibitor, and pharmaceutically acceptable salts thereof, and, (ii) after a period of at least 2 days, administering to the subject a therapeutically effective amount of an anti-cancer agent or a pharmaceutically acceptable salt thereof; wherein step (ii) treats the chemoresistant cancer in the subject.
15. The method of claim 14, wherein the period is 2 days to 2 weeks.
16. The method of claim 15, wherein the period is 2 days to 1 week.
17. The method of claim 16, wherein the period is 2 to 6 days.
18. The method of any one of claim 1, wherein the compound is chaetocin or a pharmaceutically acceptable salt thereof.
19. The method of any one of claim 1, wherein the compound is a chaetocin analogue or a pharmaceutically acceptable salt thereof.
20. A method of reversing chemoresistance of a chemoresistant cancer cell, the method comprising contacting the chemoresistant cancer cell with an effective amount of a compound selected from the group consisting of chaetocin, a chaetocin analogue, a Suv39H1/Suv39H2 inhibitor, and pharmaceutically acceptable salts thereof.
21. The method of claim 19, wherein the chaetocin analogue is a compound of formula (I): ##STR00019## or a pharmaceutically acceptable salt thereof, wherein is a single or double bond, as valency requires; each R is independently hydrogen or an optionally substituted group selected from C.sub.1-20 aliphatic, C.sub.1-20 heteroalkyl, phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring, an 8-14 membered bicyclic or polycyclic saturated, partially unsaturated or aryl ring, a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 7-14 membered bicyclic or polycyclic saturated or partially unsaturated heterocyclic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-14 membered bicyclic or polycyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or two R groups are optionally taken together with their intervening atoms to form an optionally substituted 3-14 membered, saturated, partially unsaturated, or aryl ring having, in addition to the intervening atoms, 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; R.sup.1 is R, —C(O)R, —C(O)N(R).sub.2, —S(O)R, —S(O).sub.2R, —S(O).sub.2OR, —C(R).sub.2OR, or —S(O).sub.2N(R).sub.2; and R.sup.2 is R, —[C(R).sub.2].sub.q—OR, —[C(R).sub.2].sub.q—N(R).sub.2, —[C(R).sub.2].sub.q—SR, —[C(R).sub.2].sub.q—OSi(R).sub.3, —[C(R).sub.2].sub.q—OC(O)R, —[C(R).sub.2].sub.q—OC(O)OR, —[C(R).sub.2].sub.q—OC(O)N(R).sub.2, —[C(R).sub.2].sub.q—OC(O)N(R)—SO.sub.2R or —[C(R).sub.2].sub.q—OP(OR).sub.2; or R.sup.1 and R.sup.2 are taken together with their intervening atoms to form an optionally substituted 4-7 membered heterocyclic ring having, in addition to the nitrogen atom to which R.sup.1 is attached, 0-2 heteroatoms independently selected from oxygen, nitrogen or sulfur; each q is independently 0, 1, 2, 3, or 4; R.sup.3 is an electron-withdrawing group; R.sup.4 is absent when
is a double bond, or R.sup.4 is R or halogen; R.sup.5 is absent when
is a double bond, or R.sup.5 is hydrogen or an optionally substituted C.sub.1-6 aliphatic group; n is 0, 1, 2, 3, or 4; each of R.sup.6 and R.sup.6′ is independently R, halogen, —CN, —NO.sub.2, —OR, —SR, —N(R).sub.2, —S(O).sub.2R, —S(O).sub.2N(R).sub.2, —S(O)R, —C(O)R, —C(O)OR, —C(O)N(R).sub.2, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R).sub.2, —N(R)S(O).sub.2R, or —OSi(R).sub.3; or R.sup.6 and R.sup.6′ are taken together to form ═O, ═C(R).sub.2, or ═NR; each R.sup.7 is independently R, halogen, —CN, —NO.sub.2, —OR, —OSi(R).sub.3, —SR, —N(R).sub.2, —S(O).sub.2R, —S(O).sub.2OR, —S(O).sub.2N(R).sub.2, —S(O)R, —C(O)R, —C(O)OR, —C(O)N(R).sub.2, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R).sub.2, —N(R)S(O).sub.2R, —P(R).sub.2, —P(OR).sub.2, —P(O)(R).sub.2, —P(O)(OR).sub.2, —P(O)[N(R).sub.2]2, —B(R).sub.2, —B(OR).sub.2, or —Si(R).sub.3; or two R.sup.7 are taken together with their intervening atoms to form an optionally substituted 4-7 membered ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; and R.sup.8 is —(S).sub.m—R.sup.x wherein m is 1-3 and R.sup.x is R, —SR, —C(O)R, —C(O)OR, —C(O)N(R).sub.2, —C(S)R, —S(O)R, —S(O).sub.2R, or —S(O).sub.2N(R).sub.2; and R.sup.9 is —(S).sub.p—R.sup.y, wherein p is 1-3 such that m+p is 2-4, and R.sup.y is R, —SR, —C(O)R, —C(O)OR, —C(O)N(R).sub.2, —C(S)R, —S(O)R, —S(O).sub.2R, or —S(O).sub.2N(R).sub.2; or R.sup.8 and R.sup.9 are taken together to form —S—, —(S).sub.m—[C(R).sub.2].sub.q(S).sub.p—, —(S).sub.m—(S).sub.p—, —(S).sub.m—C(O)—(S).sub.p—, —(S).sub.m—C(S)—(S).sub.p—, —(S).sub.m—S(O)—(S).sub.p—, or —(S).sub.m—S(O).sub.2—(S).sub.p—, wherein p is 1-3 such that m+p is 2-4.
22. The method of claim 21, wherein R.sup.3 is —SO.sub.2R or —COCF.sub.3.
23. The method of claim 22, wherein R.sup.3 is —SO.sub.2Ph.
24. The method of any one of claims 21 to 23, wherein R.sup.8 and R.sup.9 are taken together to form —S—, —(S).sub.m—(CH.sub.2)—(S).sub.p—, —(S).sub.m—(S).sub.p—, —(S).sub.m—C(O)—(S).sub.p—, or —(S).sub.m—C(S)—(S).sub.p—.
25. The method of any one of claims 21 to 24, wherein each of R.sup.5, R.sup.6 and R.sup.6′ is independently hydrogen.
26. The method of any one of claims 21 to 25, wherein R.sup.8 and R.sup.9 are taken together to form —S—S—.
27. The method of claim 19 or 20, wherein the chaetocin analogue is a compound of formula (II): ##STR00020## or a pharmaceutically acceptable salt thereof, wherein each R.sup.1 is independently R, —C(O)R, —C(O)N(R).sub.2, —S(O)R, —S(O).sub.2R, —S(O).sub.2OR, —C(R).sub.2OR, or —S(O).sub.2N(R).sub.2; each R is independently hydrogen or a group selected from C.sub.1-20 aliphatic, C.sub.1-20 heteroalkyl, phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring, an 8-14 membered bicyclic or polycyclic saturated, partially unsaturated or aryl ring, a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 7-14 membered bicyclic or polycyclic saturated or partially unsaturated heterocyclic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-14 membered bicyclic or polycyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or two R groups are optionally taken together with their intervening atoms to form a 3-14 membered, saturated, partially unsaturated, or aryl ring having, in addition to the intervening atoms, 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each R.sub.2 is independently R, —[C(R).sub.2].sub.q—OR, —[C(R).sub.2].sub.q—N(R).sub.2, —[C(R).sub.2].sub.q—SR, —[C(R).sub.2].sub.q—OSi(R).sub.3, —[C(R).sub.2].sub.q—OC(O)R, —[C(R).sub.2].sub.q—OC(O)OR, —[C(R).sub.2].sub.q—OC(O)N(R).sub.2, —[C(R).sub.2].sub.q—OC(O)N(R)—SO.sub.2R or —[C(R).sub.2].sub.q—OP(OR).sub.2; each q is independently 0, 1, 2, 3, or 4; each R.sup.3 is hydrogen or an electron-withdrawing group; each R.sup.5 is independently hydrogen or a C.sub.1-6 aliphatic group; each of R.sup.6 and R.sup.6′ is independently R, halogen, —CN, —NO.sub.2, —OR, —SR, —N(R).sub.2, —S(O).sub.2R, —S(O).sub.2N(R).sub.2, —S(O)R, —C(O)R, —C(O)OR, —C(O)N(R).sub.2, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R).sub.2, —N(R)S(O).sub.2R, or —OSi(R).sub.3; or R.sup.6 and R.sup.6′ are taken together to form ═O, ═C(R).sub.2 or ═NR; each n is independently 0, 1, 2, 3, or 4; each R.sup.7 is independently R, halogen, —CN, —NO.sub.2, —OR, —OSi(R).sub.3, —SR, —N(R).sub.2, —S(O).sub.2R, —S(O).sub.2OR, —S(O).sub.2N(R).sub.2, —S(O)R, —C(O)R, —C(O)OR, —C(O)N(R).sub.2, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R).sub.2, —N(R)S(O).sub.2R, —P(R).sub.2, —P(OR).sub.2, —P(O)(R).sub.2, —P(O)(OR).sub.2, —P(O)[N(R).sub.2].sub.2, —B(R).sub.2, —B(OR).sub.2, or —Si(R).sub.3; each R.sup.8 is independently —(S).sub.p—R.sup.x wherein m is 1, 2, or 3, and R.sup.x is R, —SR, —C(O)R, —C(O)OR, —C(O)N(R).sub.2, —C(S)R, —S(O)R, —S(O).sub.2R, or —S(O).sub.2N(R).sub.2; and each R.sup.9 is independently —(S).sub.p—R.sup.y wherein p is 1, 2, or 3, such that the sum of m and p is 2, 3, or 4, and R.sup.y is R, —SR, —C(O)R, —C(O)OR, —C(O)N(R).sub.2, —C(S)R, —S(O)R, —S(O).sub.2R, or —S(O).sub.2N(R).sub.2; or R.sup.8 and R.sup.9 are taken together to form —S—, —(S).sub.m—[C(R).sub.2].sub.q—(S).sub.p—, —(S).sub.m—(S).sub.p—, —(S).sub.m—C(O)—(S).sub.p—, —(S).sub.m—C(S)—(S).sub.p—, —(S).sub.m—S(O)—(S).sub.p—, or —(S).sub.m—S(O).sub.2—(S).sub.p—.
28. The method of claim 27, wherein R.sup.3 is —SO.sub.2R or —COCF.sub.3.
29. The method of claim 28, wherein R.sup.3 is —SO.sub.2Ph.
30. The method of any one of claims 27 to 29, wherein R.sup.8 and R.sup.9 are taken together to form —S—, —(S).sub.m—(CH.sub.2)—(S).sub.p—, —(S).sub.m—(S).sub.p—, —(S).sub.m—C(O)—(S).sub.p—, or —(S).sub.m—C(S)—(S).sub.p—.
31. The method of any one of claims 27 to 30, wherein each of R.sup.1, R.sup.6, and R.sup.6′ is independently hydrogen.
32. The method of any one of claims 27 to 31, wherein R.sup.8 and R.sup.9 are taken together to form —S—S—.
33. The method of claim 19 or 20, wherein the chaetocin analogue is a compound of formula (III):
ML+
D).sub.s].sub.t, (III) or a pharmaceutically acceptable salt thereof, wherein M is a cell-specific ligand unit; each L is independently a linker unit; each D independently has the structure of formula (IIIA) or (IIIB): ##STR00021## wherein each
is a single or double bond, as valency requires; each R.sup.1 is independently R, —C(O)R, —C(O)N(R).sub.2, —S(O)R, —S(O).sub.2R, —S(O).sub.2OR, —C(R).sub.2OR, or —S(O).sub.2N(R).sub.2; each R is independently hydrogen or an optionally substituted group selected from C.sub.1-20 aliphatic, C.sub.1-20 heteroalkyl, phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring, an 8-14 membered bicyclic or polycyclic saturated, partially unsaturated or aryl ring, a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 7-14 membered bicyclic or polycyclic saturated or partially unsaturated heterocyclic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and an 8-14 membered bicyclic or polycyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or two R groups are optionally taken together with their intervening atoms to form an optionally substituted 3-14 membered, saturated, partially unsaturated, or aryl ring having, in addition to the intervening atoms, 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; each R.sup.2 is independently R, —[C(R).sub.2].sub.q—OR, —[C(R).sub.2].sub.q—N(R).sub.2, —[C(R).sub.2].sub.q—SR, —[C(R).sub.2].sub.q—OSi(R).sub.3, —[C(R).sub.2].sub.q—OC(O)R, —[C(R).sub.2].sub.q—OC(O)OR, —[C(R).sub.2].sub.q—OC(O)N(R).sub.2, —[C(R).sub.2].sub.q—OC(O)N(R)—SO.sub.2R or —[C(R).sub.2].sub.q—OP(OR).sub.2; or R.sup.1 and R.sup.2 are taken together with their intervening atoms to form an optionally substituted 4-7 membered heterocyclic ring having, in addition to the nitrogen atom to which R.sup.1 is attached, 0-2 heteroatoms independently selected from oxygen, nitrogen, and sulfur; each q is independently 0, 1, 2, 3, or 4; each R.sub.3 is independently R or an electron-withdrawing group; each R.sup.4 is independently absent, when
is a double bond, or is independently R or halogen; each R.sup.5 is independently absent, when
is a double bond, or is independently hydrogen or an optionally substituted C.sub.1-6 aliphatic group; each of R.sup.6 and R.sup.5 is independently R, halogen, —CN, —NO.sub.2, —OR, —SR, —N(R).sub.2, —S(O).sub.2R, —S(O).sub.2N(R).sub.2, —S(O)R, —C(O)R, —C(O)OR, —C(O)N(R).sub.2, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R).sub.2, —N(R)S(O).sub.2R, or —OSi(R).sub.3; or R.sup.6 and R.sup.6′ are taken together to form ═O, ═C(R).sub.2, or ═NR; each n is independently 0, 1, 2, 3, or 4; each R.sup.7 is independently R, halogen, —CN, —NO.sub.2, —OR, —OSi(R).sub.3, —SR, —N(R).sub.2, —S(O).sub.2R, —S(O).sub.2OR, —S(O).sub.2N(R).sub.2, —S(O)R, —C(O)R, —C(O)OR, —C(O)N(R).sub.2, —C(O)N(R)—OR, —N(R)C(O)OR, —N(R)C(O)N(R).sub.2, —N(R)S(O).sub.2R, —P(R).sub.2, —P(OR).sub.2, —P(O)(R).sub.2, —P(O)(OR).sub.2, —P(O)[N(R).sub.2].sub.2, —B(R).sub.2, —B(OR).sub.2, or —Si(R).sub.3; or two R.sup.7 are taken together with their intervening atoms to form an optionally substituted 4-7 membered ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur; each R.sup.8 is independently —(S).sub.m—R.sup.x wherein m is 1, 2, or 3, and R.sup.x is R, —SR, —C(O)R, —C(O)OR, —C(O)N(R).sub.2, —C(S)R, —S(O)R, —S(O).sub.2R, or —S(O).sub.2N(R).sub.2; each R.sup.9 is independently —(S).sub.p—R.sup.y wherein p is 1, 2, or 3, such that the sum of m and p is 2, 3, or 4, and R is R, —SR, —C(O)R, —C(O)OR, —C(O)N(R).sub.2, —C(S)R, —S(O)R, —S(O).sub.2R, or —S(O).sub.2N(R).sub.2; or R.sup.8 and R.sup.9 are taken together to form —S—, —(S).sub.m—[C(R).sub.2].sub.q—(S).sub.p—, —(S).sub.m—(S).sub.p—, —(S).sub.m—C(O)—(S).sub.p—, —(S).sub.m—C(S)—(S).sub.p—, —(S).sub.m—S(O)—(S).sub.p—, or —(S).sub.m—S(O).sub.2—(S).sub.p—; s is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and t is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; provided that one of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.6′, R.sup.7, R.sup.8, and R.sup.9 is substituted with a bond to L.
34. The method of claim 33, wherein the chaetocin analogue is a compound of formula (IIIA): ##STR00022## or a pharmaceutically acceptable salt thereof.
35. The method of claim 33, wherein the chaetocin analogue is a compound of formula (IIIB): ##STR00023## or a pharmaceutically acceptable salt thereof.
36. The method of any one of claims 33 to 35, wherein s is 1.
37. The method of any one of claims 33 to 36, wherein L is self-immolative.
38. The method of any one of claims 33 to 37, wherein M is an antibody.
39. The method of any one of claims 33 to 38, wherein R.sup.3 is R.
40. The method of any one of claims 33 to 38, wherein R.sup.3 is an electron-withdrawing group.
41. The method of any one of claims 33 to 38, wherein R.sup.3 is —S(O).sub.2R, —S(O).sub.2—[C(R).sub.2].sub.q—R, —S(O).sub.2—[C(R).sub.2].sub.q—B(OR).sub.2, —S(O).sub.2—[C(R).sub.2].sub.q—Si(R).sub.3, —S(O).sub.2OR, —S(O).sub.2N(R).sub.2, —S(O)R, —C(O)R, —C(O)OR, —C(O)N(R).sub.2, —C(O)N(R)—OR, —P(O)(R).sub.2, —P(O)(OR).sub.2, or —P(O)[N(R).sub.2].sub.2.
42. The method of any one of claims 33 to 38, wherein R.sup.3 is —SO.sub.2R.
43. The method of any one of claims 33 to 42, wherein each R.sup.4 is independently an optionally substituted group selected from phenyl, an 8-14 membered bicyclic or tricyclic aryl ring, a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and an 8-14 membered bicyclic or tricyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
44. The method of any one of claims 33 to 43, wherein R.sup.8 is —(S).sub.m—R.sup.x, wherein m is 1, 2, or 3, and R.sup.x is —SR, —C(O)R, —C(O)OR, —C(O)N(R).sub.2, —C(S)R, —S(O)R, —S(O).sub.2R, or —S(O).sub.2N(R).sub.2; and R.sup.9 is —(S).sub.p—R.sup.y wherein p is 1, 2, or 3, such that the sum of m and p is 2, 3, or 4, and R.sup.y is —SR, —C(O)R, —C(O)OR, —C(O)N(R).sub.2, —C(S)R, —S(O)R, —S(O).sub.2R, or —S(O).sub.2N(R).sub.2; or R.sup.8 and R.sup.9 are taken together to form —S—, —(S).sub.m—[C(R).sub.2].sub.q—(S).sub.p—, —(S).sub.m—(S).sub.p—, —(S).sub.m—C(O)—(S).sub.p—, —(S).sub.m—C(S)—(S).sub.p—, —(S).sub.m—S(O)—(S).sub.p—, or —(S).sub.m—S(O).sub.2—(S).sub.p—.
45. The method of any one of claims 33 to 43, wherein R.sup.8 and R.sup.9 are taken together to form —S—, —(S).sub.m—C(R).sub.2—(S).sub.p—, —(S).sub.m—(S).sub.p—, —(S).sub.m—C(O)—(S).sub.p—, —(S).sub.m—C(S)—(S).sub.p—, —(S).sub.m—S(O)—(S).sub.p—, or —(S).sub.m—S(O).sub.2—(S).sub.p—.
46. The method of any one of claims 33 to 43, wherein: R.sup.8 is —(S).sub.m—R.sup.x wherein m is 1 and R.sup.x is —SR or —C(O)R; and R.sup.9 is —(S).sub.p—R.sup.y wherein p is 1 and R.sup.y is —SR or —C(O)R.
47. The method of any one of claims 33 to 43, wherein R.sup.8 and R.sup.9 are taken together to form —(S).sub.m—(S).sub.p—, —S—C(O)—S—, or —S—C(S)—S—.
48. The method of any one of claims 33 to 47, wherein: each is independently a single bond; each R.sup.4 is independently R or halogen; and each R.sup.5 is independently hydrogen or an optionally substituted C.sub.1-8 aliphatic group.
49. The method of claim 19, wherein the chaetocin analogue is a compound selected from the group consisting of: ##STR00024## and pharmaceutically acceptable salt thereof.
50. The method of claim 1, wherein the chemoresistant cancer has an epithelial-to-mesenchymal transition (EMT) pathway implicated.
51. The method of claim 1, wherein the chemoresistant cancer is a chemoresistant glioblastoma, breast cancer, lung cancer, or blood cancer.
52. The method of claim 51, wherein the chemoresistant cancer is a chemoresistant breast cancer, kidney cancer, or lung cancer.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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[0334] Epigenetic changes are those which the phenotypic change induced upon transient exposure to a signal (e.g. chaetocin) persists stably even in the absence of the inducing signal.
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[0339] Because chaetocin has been shown as a specific inhibitor of constitutive heterochromatin proteins SUV39H1 and SUV39H2, we asked whether a transient knockdown of different constitutive heterochromatin proteins also causes an epigenetic reversal of mesenchymal phenotype.
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DETAILED DESCRIPTION
[0355] Generally, the invention provides methods for treating cancer, e.g., by addressing the problem of cancer chemoresistance. In particular, the invention provides methods of treatment of cancer (e.g., chemoresistant cancer or mesenchymal cancer (e.g., a chemoresistant mesenchymal cancer)) in a subject (e.g., a mammalian subject, such as a human) in need thereof by administering chaetocin, a chaetocin analogue, a Suv39H1/Suv39H2 inhibitor, or a pharmaceutically acceptable salt thereof prior to the administration of an anti-cancer agent (e.g., a chemotherapeutic agent). In some embodiments, the methods of the invention may be used in the treatment of a mesenchymal cancer. In some embodiments, the methods of the invention may be used in the treatment of a chemoresistant cancer. The invention also provides methods of killing cancer cells (e.g., chemoresistant cancer cells or mesenchymal cancer cells (e.g., chemoresistant mesenchymal cancer cells)). The invention also provides methods of reversing chemoresistance and killing of chemoresistant cancer cells.
[0356] Without wishing to be bound by theory, it is believed that the compounds disclosed herein elicit an epigenetic change converting mesenchymal, chemotherapy-resistant cancer cells to an epithelial, chemotherapy-sensitive phenotype. It is believed that this reversal is responsible, at least in part, for the restoration of chemotherapy sensitivity in cancer cells. A cancer may become mesenchymal in response to chemotherapy or other stresses, or as the cancer progresses and metastasizes converting some cancer cells into a mesenchymal state. Preferably, the cancers (e.g., chemoresistant cancers) treated using methods disclosed herein include a mesenchymal cancer cell (e.g., a chemoresistant, mesenchymal cancer cell). Also, preferably, the chemoresistant cancer cells are chemoresistant mesenchymal cancer cells.
Pharmaceutical Compositions
[0357] The compounds used in the methods described herein are preferably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo. Pharmaceutical compositions typically include a compound as described herein and a pharmaceutically acceptable excipient.
[0358] A pharmaceutical composition can be formulated to be compatible with its intended route of administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
[0359] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
[0360] Sterile injectable solutions can be prepared by incorporating the composition in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the composition into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
[0361] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
[0362] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
[0363] The compositions can be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.
[0364] Pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. Methods of formulating pharmaceutical agents are known in the art. The compositions described herein can be administered locally, e.g., to the site of a cancer in a subject. Examples of local administration include epicutaneous, inhalational (e.g. byway of a nebulizer), intra-articular, intrathecal, intravaginal, intravitreal, intrauterine, intra-lesional administration, lymph node administration, intratumoral administration and administration to a mucous membrane of the subject, wherein the administration is intended to have a local and not a systemic effect. As an example, for the treatment of a cancer described herein, the compositions described herein may be administered locally (e.g., intratumorally) in a compound-impregnated substrate such as a wafer, microcassette, or resorbable sponge placed in direct contact with the affected tissue. Alternatively, the composition is infused into the brain or cerebrospinal fluid using standard methods. A composition for use in the methods described herein can be administered at the site of a tumor, e.g., intratumorally. In certain embodiments, the agent is administered to a mucous membrane of the subject.
Preparation of Compounds
[0365] Compounds disclosed herein may be prepared using methods and techniques known in the art. Non-limiting examples of the search strategies may be found, e.g., in: (a) P. W. Trown, Biochem. Biophys. Res. Commun., 1968, 33, 402; (b) T. Hino and T. Sato, Tetrahedron Lett., 1971, 12, 3127; (c) H. Poisel and U. Schmidt, Chem. Ber., 1971, 104, 1714; (d) H. Poisel and U. Schmidt, Chem. Ber., 1972, 105, 625; (e) E. Ohler, F. Tataruch and U. Schmidt, Chem. Ber., 1973, 106, 396; (f) H. C. J. Ottenheijm, J. D. M. Herscheid, G. P. C. Kerkhoff and T. F. Spande, J. Org. Chem., 1976, 41, 3433; (g) D. L. Coffen, D. A. Katonak, N. R. Nelson and F. D. Sancilio, J. Org. Chem., 1977, 42, 948; (h) J. D. M. Herscheid, R. J. F. Nivard, M. W. Tijhuis, H. P. H. Scholten and H. C. J. Ottenheijm, J. Org. Chem., 1980, 45, 1885; (i) R. M. Williams, R. W. Armstrong, L. K. Maruyama, J.-S. Dung and O. P. Anderson, J. Am. Chem. Soc., 1985,107,3246; (j) C. J. Moody, A. M. Z. Slawin and D. Willows, Org. Biomol. Chem., 2003, 1, 2716; (k) A. E. Aliev, S. T. Hilton, W. B. Motherwell and D. L. Selwood, Tetrahedron Lett., 2006, 47, 2387; (l) L. E. Overman and T. Sato, Org. Lett., 2007, 9, 5267; (m) N. W. Polaske, R. Dubey, G. S. Nichol and B. Olenyuk, Tetrahedron: Asym., 2009, 20, 2742; (n) B. M. Ruff, S. Zhong, M. Nieger and S. Brase, Org. Biomol. Chem., 2012, 10, 935; (o) K. C. Nicolaou, D. Giguere, S. Totokotsopoulos and Y.-P. Sun, Angew. Chem. Int. Ed., 2012, 51, 728; for selected epidithiodiketopiperazine total syntheses, see: (a) Y. Kishi, T. Fukuyama and S. Nakatsuka, J. Am. Chem. Soc., 1973, 95, 6492; (b) Y. Kishi, S. Nakatsuka, T. Fukuyama and M. Havel, J. Am. Chem. Soc., 1973, 95, 6493; (c) T. Fukuyama and Y. Kishi, J. Am. Chem. Soc., 1976, 98, 6723; (d) R. M. Williams and W. H. Rastetter, J. Org. Chem., 1980, 45, 2625; (e) G. F. Miknis and R. M. Williams, J. Am. Chem. Soc., 1993, 115, 536; (f) E. Iwasa, Y. Hamashima, S. Fujishiro, E. Higuchi, A. Ito, M. Yoshida and M. Sodeoka, J. Am. Chem. Soc., 2010, 132, 4078; (g) J. E. DeLorbe, S. Y. Jabri, S. M. Mennen, L. E. Overman and F.-L. Zhang, J. Am. Chem. Soc., 2011, 133, 6549; (h) K. C. Nicolaou, S. Totokotsopoulos, D. Giguere, Y.-P. Sun and D. Sarlah, J. Am. Chem. Soc., 2011, 133, 8150; (i) J. A. Codelli, A. L. A. Puchlopek and S. E. Reisman, J. Am. Chem. Soc., 2012, 134, 1930; for other synthetic strategies relevant to epipolythiodiketopiperazines, see: (a) J. Kim, J. A. Ashenhurst and M. Movassaghi, Science, 2009, 324, 238; (b) J. Kim and M. Movassaghi, J. Am. Chem. Soc., 2010, 132, 14376).
Regimens
[0366] Timing of Administration
[0367] In the methods disclosed herein, a chemoresistant cancer cell may be killed by contacting the chemoresistant cancer cell with an effective amount of chaetocin, a chaetocin, analogue, a Suv39H1/Suv39H2 inhibitor, or a pharmaceutically acceptable salt thereof, and after a period of at least 5 hours (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours or more), contacting the cell with an anti-cancer agent or a pharmaceutically acceptable salt thereof, thereby killing the chemoresistant cancer cell.
[0368] The invention further provides methods of treating a chemoresistant cancer in a subject in need thereof (e.g., a mammalian subject, such as a human) by administering to the subject a therapeutically effective amount of chaetocin, a chaetocin analogue, a Suv39H1/Suv39H2 inhibitor, or a pharmaceutically acceptable salt thereof, and after a period of at least 5 hours (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours or more), administering to the subject a therapeutically effective amount of an anti-cancer agent or a pharmaceutically acceptable salt thereof, thereby treating the chemoresistant cancer in the subject.
[0369] In some embodiments, the period is 5 hours to 2 weeks (e.g., for the methods of killing a chemoresistant cancer cell). For example, the period may be 5 hours to 2 weeks, 6 hours to 2 weeks, 9 hours to 2 weeks, 12 hours to 2 weeks, 18 hours to 2 weeks, 24 hours to 2 weeks, 2 days to 2 weeks, 3 days to 2 weeks, 4 days to 2 weeks, 5 days to 2 weeks, 6 days to 2 weeks, 7 days to 2 weeks, 8 days to 2 weeks, 9 days to 2 weeks, 10 days to 2 weeks, 11 days to 2 weeks, 12 days to 2 weeks, or 13 days to 2 weeks.
[0370] In some embodiments, the period is 2 days to 2 weeks (e.g., for the methods of treating a chemoresistant cancer). For example, the period may be 2 days to 2 weeks, 3 days to 2 weeks, 4 days to 2 weeks, 5 days to 2 weeks, 6 days to 2 weeks, 7 days to 2 weeks, 8 days to 2 weeks, 9 days to 2 weeks, 10 days to 2 weeks, 11 days to 2 weeks, or 13 days to 2 weeks.
[0371] The treatment regimen described herein can treat chemoresistant cancer by increasing cancer cell death in a subject (e.g., a mammalian subject, such as a human) or in a cancer cell culture (e.g., a culture generated from a patient tumor sample, a cancer cell line, or a repository of patient samples). Treatment can increase cancer cell death by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to before treatment to a subject or cancer cell culture. Treatment can increase cancer cell death in a subject or cancer cell culture between 5-20%, between 5-50%, between 10-50%, between 20-80%, between 20-70%. The treatment can also act to inhibit cancer cell growth, proliferation, metastasis, migration, or invasion. Cancer cell growth, proliferation, metastasis, migration, or invasion can be decreased in the subject or cancer cell culture at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, compared to before the treatment. Cancer cell growth, proliferation, metastasis, migration, or invasion can be decreased in the subject or cancer cell culture between 5-20%, between 5-50%, between 10-50%, between 20-80%, between 20-70%.
[0372] The methods described herein may act to alter (e.g., increase or decrease) the expression level and/or activity of an mRNA or protein of a gene associated with the cancer. For example, the compositions disclosed herein may increase/decrease the expression level and/or activity of chromatin-modifying proteins by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more. In some embodiments, the chromatin regulator protein is Suppressor of Variegation 3-9 Homolog 1. In some embodiments, the chromatin regulator protein is Suppressor of Variegation 3-9 Homolog 2. Expression levels and or activity levels may be measured using standard methods known in the art such as, e.g., western blots, immunohistochemistry, immunoprecipitation, qRT-PCR, in situ hybridization, ELISA assay, among others.
[0373] Routes of Administration
[0374] Therapeutic agents used in the methods described herein may be administered to a subject in need thereof by standard methods. For example, the composition can be administered by any of a number of different routes including, e.g., systemic administration such as intravenous, intraperitoneal, intradermal, subcutaneous, percutaneous injection, oral, intranasal, transdermal (topical), or transmucosal. The composition can be administered orally or administered by injection, e.g., intramuscularly, intravenously, intraperitoneally, intrathecally, intracerebroventricularly, intraparenchymally, or intratumorally. In some embodiments, the composition is administered intratumorally. The most suitable route for administration in any given case will depend on the particular agent administered, the patient, the particular disease or condition being treated, pharmaceutical formulation methods, administration methods (e.g., administration time and administration route), the patient's age, body weight, sex, severity of the diseases being treated, the patient's diet, and the patient's excretion rate. The agent can be encapsulated or injected, e.g., in a viscous form, for delivery to a chosen site, e.g., a tumor site. The agent can be provided in a matrix capable of delivering the agent to the chosen site. Matrices can provide slow release of the agent and provide proper presentation and appropriate environment for cellular infiltration. Matrices can be formed of materials presently in use for other implanted medical applications. The choice of matrix material is based on any one or more of: biocompatibility, biodegradability, mechanical properties, and cosmetic appearance and interface properties. One example is a collagen matrix.
[0375] The compositions described herein can be incorporated into pharmaceutical compositions suitable for administration to a subject, e.g., a human. Such compositions typically include the agent and a pharmaceutically acceptable carrier. As used herein the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances are known. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention. Supplementary active compounds can also be incorporated into the compositions.
[0376] Anti-Cancer Agents
[0377] i. Chemotherapeutic Agents
[0378] Chemotherapeutic agents suitable for use with the methods described herein include alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxins, L-asparaginase, topoisomerase inhibitors, interferons, platinum coordination complexes, anthracenedione substituted urea, methyl hydrazine derivatives, adrenocortical suppressant, adrenocorticosteroides, progestins, estrogens, antiestrogen, androgens, antiandrogen, and gonadotropin-releasing hormone analogue. Non-limiting examples of chemotherapeutic agents include alkylating agents (e.g., cisplatin, carboplatin, oxalipiatin, mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil, or ifosfamide), antimetabolites (e.g., fluorouracil (5-FU), gemcitabine, methotrexate, cytosine arabinoside, fludarabine, and floxuridine), antimitotics (e.g., a taxane (e.g., paclitaxel or decetaxel) or a vinca alkaloid (e.g., vincristine, vinblastine, vinorelbine, or vindesine)), anthracyclines (e.g., doxorubicin, daunorubicin, valrubicin, idarubicin, epirubicin, or an actinomycin (e.g., actinomycin D)), cytotoxic antibiotics (e.g., mitomycin, plicamycin, or bleomycin), topoisomerase inhibitors (e.g., a camptothecin (e.g., irinotecan or topotecan) or an epipodophyllotoxin derivative (e.g., amsacrine, etoposide, or teniposide), and pharmaceutically acceptable salts thereof.
[0379] One of skill in the art would recognize that other chemotherapeutic agents may also be used with the methods of the invention.
[0380] ii. Checkpoint Inhibitors
[0381] One type of agent that can be also administered in combination with the methods described herein is a checkpoint inhibitor. One having skill in the art would be able to select a known checkpoint inhibitor suitable for treatment of a particular chemoresistant cancer in conjunction with the methods described herein.
[0382] iii. Non-Drug Modalities
[0383] Another type of therapeutic modality that can be administered in conjunction with the methods disclosed herein is a therapeutic modality that is a non-drug treatment. For example, the therapeutic modality may be radiation therapy, cryotherapy, alternating electric field therapy, hyperthermia and/or surgical excision of tumor tissue
[0384] iv. Biologic Anti-Cancer Agents
[0385] Another type of therapeutic agent that can be administered in conjunction with the methods described herein is an anti-cancer agent that is a biologic, such as a cytokine (e.g., interferon or an interleukin) used in cancer treatment. In other embodiments, the biologic is an anti-angiogenic agent, such as an anti-VEGF agent. In some embodiments, the biologic is an immunoglobulin-based biologic, such as, for example, a monoclonal antibody (e.g., a humanized antibody, a fully human antibody, an Fc fusion protein, or a functional fragment thereof) that agonizes a target to stimulate an anti-cancer response, or antagonizes an antigen important for cancer. Biologic anti-cancer agents are known in the art.
[0386] Cancers
[0387] In the methods described herein, the cancer may be any solid or liquid cancer and includes benign or malignant tumors, and hyperplasias. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is kidney cancer. In some embodiments, the cancer is lung cancer. Other cancers suitable for treatment using the methods described herein include gastrointestinal cancer (such as non-metastatic or metastatic colorectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular cancer, cholangiocellular cancer, oral cancer, lip cancer); urogenital cancer (such as hormone sensitive or hormone refractory prostate cancer, renal cell cancer, bladder cancer, penile cancer); gynecological cancer (such as ovarian cancer, cervical cancer, endometrial cancer); lung cancer (such as small-cell lung cancer and non-small-cell lung cancer); head and neck cancer (e.g., head and neck squamous cell cancer); CNS cancer including malignant glioma, astrocytomas, retinoblastomas and brain metastases; malignant mesothelioma; non-metastatic or metastatic breast cancer (e.g., hormone refractory metastatic breast cancer); skin cancer (such as malignant melanoma, basal and squamous cell skin cancers, Merkel Cell Carcinoma, lymphoma of the skin, Kaposi Sarcoma); thyroid cancer; bone and soft tissue sarcoma; and hematologic neoplasias (such as multiple myeloma, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, Hodgkin's lymphoma). Preferably, a chemoresistant cancer is a chemoresistant glioblastoma, breast cancer, lung cancer, or blood cancer.
[0388] Subjects who can be treated with the methods disclosed herein include subjects who have had one or more tumors resected, received chemotherapy or other pharmacological treatment for the cancer, received radiation therapy, and/or received other therapy for the cancer. Subjects who have not previously been treated for cancer can also be treated with the methods disclosed herein.
[0389] In some embodiments, the compounds described herein may be administered in an amount and for a time effective to result in one of (or more, e.g., 2 or more, 3 or more, 4 or more of): (a) reduced tumor size, (b) reduced rate of tumor growth, (c) increased tumor cell death (d) reduced tumor progression, (e) reduced number of metastases, (f) reduced rate of metastasis, (g) reduced tumor migration, (h) reduced tumor invasion, (i) reduced tumor volume, (j) decreased tumor recurrence, (k) increased survival of subject, (l) increased progression free survival of subject.
[0390] The methods described herein may include a step of selecting a treatment for a patient. The method includes (a) identifying (e.g., diagnosing) a patient who has cancer or is at risk of developing cancer, and treating the condition in the patient. In some embodiments, the method includes administering the selected treatment to the subject. In some embodiments, a patient is identified as having cancer based on imaging (e.g., MRI, CT, or PET scan), biopsy, or blood sample (e.g., detection of blood antigen markers, circulating tumor DNA (e.g., by PCR).
[0391] The method may also include a step of assessing the subject for a parameter of cancer progression or remission, e.g., assessing the subject for one or more (e.g., 2 or more, 3 or more, 4 or more) of: primary tumor size (e.g., by imaging), number of metastases (e.g., by imaging or biopsy), cell death in situ (e.g., by biopsy), blood antigen markers (e.g., by ELISA), circulating tumor DNA (e.g., by PCR), or function of the affected organ (e.g., by a test of circulating enzymes for liver, albuminuria for kidney, lung capacity for lung, etc.).
[0392] Preferably, the chemoresistant cancer are all cancers which are mesenchymal in nature, or individual cancer cells which are mesenchymal or become mesenchymal in response to chemotherapy or other stresses, or as the cancer progresses and metastasizes generating mesenchymal cancer cells. Any of the above-listed cancers may be a chemoresistant cancer including chemoresistant, mesenchymal cancer cells.
[0393] Epithelial-to-Mesenchymal Transition
[0394] Without wishing to be bound by theory, cancer cells may acquire chemotherapeutic resistance by undergoing an epithelial-to-mesenchymal transition (EMT) through which cells acquire mesenchymal migratory properties and enhance their radiation and chemotherapy resistance as well as their tumor-initiating potential. This process is often accompanied by acquisition of stem-cell like properties among cancer cells (e.g., cancer stem cells). The key inducers of EMT are a set of transcription factors, including Snail Family Transcriptional Repressor 1 (SNAIL) and Twist Family BHLH Transcription Factor 1 (TWIST), which reprogram epithelial cells into a mesenchymal fate, increasing their resistance to a variety of stresses, including chemotherapy. EMT is critical for cancer resistance and metastasis. It is one of the most important targets for cancer therapy for which few drugs exist. The compounds, pharmaceutical compositions, and methods described herein may be used as an effective treatment in reversing chemoresistance in cancer by reversing EMT in chemoresistant cancers and/or cancer cells. Without wishing to be bound by theory, this process may convert mesenchymal, TIC-rich, chemotherapy-resistant cancer cells into an epithelial, TIC-depleted, chemotherapy-sensitive cancer.
EXAMPLES
[0395] The following are various exemplary compositions and methods which describe the invention. It is understood that other embodiments may be practiced given the general description provided above.
Example 1. Mesenchymal Cancer Stem Cells are Sensitive to a Heterochromatin Inhibitor
[0396] Previous work in the fission yeast revealed that heterochromatin proteins play an essential role in establishing an adaptive transcriptional program, permitting cells to resist stress in reversible dormancy. To determine whether constitutive heterochromatin proteins are similarly required for the development of chemotherapy in cancer, the efficacy of a heterochromatin inhibitor, which selectively represses the activities of two proteins critical for constitutive heterochromatin formation, namely suppressor of variegation 3-9 homolog 1 (Suv39H1) and suppressor of variegation 3-9 homolog 2 (Suv39H2), was tested in human cells, using 2-D, spheroid and tumorsphere assays. To determine the sensitivity of well-established mesenchymal and epithelial cancer cells to a heterochromatin inhibitor, chaetocin, the degree of apoptotic cell death was measured in two cancer cell lines, MDA-MB-231 and MDA-MB-468. MDA-MB-231 is a well-established breast cancer cell line which is rich for the so called ‘cancer stem cells’ or (CSC). Another term used for these cells is ‘tumor-initiating cells’ (TIC), which is generally accepted as a more accurate term for describing these cells. These cells have a high propensity to initiate a tumor compared to their non-TIC cells, such as MDA-MB-468 cells. Under separate culture conditions, both cancer cell lines were exposed to increasing doses of chaetocin (0 nM, 100 nM, and 200 nM) and apoptosis was measured by assaying cleaved caspase 3, a marker of apoptotic cell death. As shown in
[0397] To test if other mesenchymal, TIC-rich, chemotherapy resistant cell lines of different tissue origin (e.g. kidney) are also sensitive to chaetocin treatment, normal and cancerous breast (control) and renal cells were treated in culture with 100 nM chaetocin for 1 day, after which cell growth was assayed using a proliferation assay (
[0398] To determine whether sensitivity of cancer cells to chaetocin correlates with mesenchymal cell phenotype, the sensitivity or resistance of 800 cancer cell lines to chaetocin was assayed by the group of Cyril Benes treating each cell line with chaetocin at concentrations of 0.005 μM, 0.01 μM, 0.02 μM, 0.04 μM, 0.08 μM, 0.16 μM, 0.32 μM, 0.64 μM, and 1.28 μM. Cell viability was measured several days following treatment. Because the transcriptomes of the cell lines are available, a gene set enrichment analysis (GSEA) was performed in which cells were ranked based on their mesenchymal gene expression signature (y-axis) versus sensitivity to chaetocin (x-axis)
[0399] Tables 1-2 show the results of elastic net (EN) regression, a penalized linear modelling technique, to identify cooperative interactions among multiple transcripts which correlate with sensitivity (Table 1) or resistance (Table 2) to chaetocin treatment.
TABLE-US-00001 TABLE 1 Elastic net regression analysis of chaetocin sensitivity Gene Frequency Effect NREP 1 −0.4950443 ZEB2 0.99 −0.2174459 ARHGEF6 0.94 −0.0887254 TMEM131L 0.92 −0.0806591 MEX3B 0.75 −0.0538461 AFF3 0.72 −0.0395303 FBXL7 0.69 −0.0356776 PLEKHO1 0.68 −0.0337116 FOXN3 0.68 −0.0297147 ZNF549 0.64 −0.0242701 KATNAL1 0.64 −0.0216114 HMGXB4 0.48 −0.0155139 TTBK2 0.57 −0.0145324 TSHZ3 0.57 −0.0144108 TNPO2 0.42 −0.006318 SLC16A2 0.18 −0.0040996 RNF122 0.33 −0.0036895 VAMP4 0.33 −0.0035611 NIN 0.33 −0.0030086 MAGEB10 0.18 −0.0020914 GFRA2 0.16 −0.0013702 JARID2 0.07 −0.0009996 SEPT6 0.03 −0.0009556 ADGRA2 0.03 −0.0008249 GPC2 0.03 −0.0008043 ESS2 0.07 −0.0007965 ZNF136 0.07 −0.0007581 IFFO1 0.03 −0.000747 PI4KA 0.07 −0.0007168 PBRM1 0.03 −0.0006938 EVL 0.03 −0.0006415 LIMD2 0.03 −0.0006163 JAM2 0.03 −0.0005928 RNF150 0.03 −0.0005375 TCF4 0.03 −0.0004947 PTBP2 0.03 −0.0004588 ZNF439 0.03 −0.0004488 RIMS3 0.03 −0.0004462 TNRC6C 0.03 −0.0004301 MAP3K3 0.03 −0.0004246 EIF3G_CN 0.03 −0.0004179 FAM212A 0.03 −0.000386 SAP30 0.03 −0.0003717 AP1M1 0.03 −0.0003477 METTL14 0.03 −0.000329 EBF1 0.03 −0.0003269 SMARCA4 0.03 −0.0003253
TABLE-US-00002 TABLE 2 Elastic net regression analysis of chaetocin resistance Gene Name Frequency Effect PPARG 0.03 0.00094769 AKR1C2 0.03 0.00098325 DHCR7 0.18 0.00156451 PPAP2C 0.24 0.00249999 VWDE 0.18 0.00279531 PNO1 0.33 0.0039486 SRXN1 0.5 0.00445076 FERMT1 0.61 0.00569307 SGK2 0.46 0.00693698 EPCAM 0.5 0.00711634 SLC35F2 0.35 0.00758156 CYP2S1 0.44 0.00913731 ECI1 0.46 0.00961131 C11orf54 0.48 0.01157074 LSR 0.63 0.01684451 FTL 0.49 0.01978019 ZDHHC23 0.61 0.0251087 CMTM4 0.67 0.0259746 SH3BGRL2 0.68 0.02649864 MROH9 0.54 0.02853239 KLF5 0.68 0.03227002 CYSTM1 0.7 0.03510757 TXNRD1 0.7 0.038292 SYNGR2 0.74 0.04247359 AKT1S1 0.75 0.05141428 CYP4F11 0.85 0.05607174 EID3 0.83 0.08004353 CMTM8 0.89 0.08562629 NQO1 0.96 0.08968791 GDE1 0.94 0.09767863 KRT8 0.96 0.10380447 ABCC3 1 0.1175382 MGAT4B 0.97 0.13354118 BAIAP2L1 1 0.14460199 UGDH 1 0.15593392 HTATIP2 0.98 0.15980576 LIPH 1 0.17505171 AGPAT9 1 0.19069813 GCLC 1 0.19122061 SLC7A11 0.98 0.19141043 F2RL1 1 0.19283263 SQSTM1 1 0.21184296 GPX2 1 0.21406466 TLCD1 1 0.22138448 FZD5 1 0.23072741 KRT18 1 0.23216695 RPE 1 0.36878775
TABLE-US-00003 TABLE 3 Summary of elastic net regression analysis focusing on chromatin genes which contribute to sensitivity Gene Name Effect −0.2174459
−0.0297147
−0.0144108
−0.0009996 ZNF136 −0.0007581
−0.0003717
−0.0003253 ZNF85 −0.0002408
−0.0001952 BEND5 −0.0001146
−0.0006938
−6.32E−05
−3.68E−05 BRD1 −4.06E−05
−1.44E−05
−8.75E−06 JAZF1 −3.17E−06
[0400] In Tables 1-3, Gene: the name of the gene in EN regression model; Frequency: 100 modeling iterations were performed and the frequency at which each transcript is present in the resulting model is reported (e.g. a frequency of 1 indicates that the feature was present in all 100 models); and Effect: Strength of the association between a gene and chaetocin response. Effect <0: Sensitizing feature. Effect >0 resistant-inducing feature. Bolded genes correspond to those encoding proteins involved in chromatin regulation. Italicized letters correspond to chromatin-regulatory genes that have been shown to be involved in EMT.
[0401] Gene ontology analysis revealed that chaetocin sensitivity correlated with a set of 16 gene clusters, encompassing genes involved in transcriptional co-repression, chromatin regulation, DNA-binding via zinc finger proteins, mental retardation, chromatin/neurogenesis, and transcription. The same analysis performed with respect to chaetocin resistance revealed 30 gene clusters, encompassing oxidation/reduction processes, NADP binding, reactive oxygen species response, tight junctions, and actin binding, among others. Table 3 is the results of EN analysis focusing only one chromatin regulators. Together these EN regression analyses reveal that expression of several chromatin-regulatory proteins with previously identified roles in EMT correlate with chaetocin sensitivity.
[0402] To determine whether chaetocin affects TIC death in a tumor-like environment, 3-D tumor spheroid assays were performed in the presence or absence of chaetocin. Tumor spheroids are a model of a real tumor, as they form a 3-D cell mass similar to tumors in vivo. The local microenvironment of a spheroid recapitulates many of the in vivo properties of tumor. Importantly, the hypoxic and often nutrient depleted microenvironment at the center of the tumor is favorable for converting epithelial, non-TIC, chemotherapy-sensitive cancer cells into mesenchymal, TIC-rich, chemotherapy-resistant cells. In this assay, cells were grown on 2-dimensional plates for two days and then transferred to the spheroid system showed in
[0403] To further determine the effect of chaetocin on eliminating TICs, a tumorsphere assay was performed on cancer cells under increasing concentrations of chaetocin. A tumorsphere is a solid spherical formation developed from the proliferation of one cancer stem/progenitor cell. These tumorspheres are easily distinguishable from single or aggregated cells as the cells appear to become fused together and individual cells cannot be identified. The size of the tumorspheres vary from less than 50 micrometers to 250 micrometers, and number of tumorspheres formed was used to characterize the cancer stem/progenitor cell population within a population of in vitro cultured cancer cells. Cells were enriched for TICs and grown in serum-free, non-adherent conditions in order to enrich the cancer stem/progenitor cell population as only cancer stem/progenitor cells can survive and proliferate in this environment.
Example 2. Increased Expression of Mesenchymal Markers Predicts Poor Outcomes in Cancer Patients
[0404] To determine whether increased expression of mesenchymal transcription factors predicts poor patient outcomes in cancer, the survival rates of patients stratified by high or low expression of mesenchymal transcription factors (TF) SNAI1 (snail family transcriptional repressor 1), SNAI2 (snail family transcriptional repressor 2), TWIST1 (twist family BHLH transcription factor 1), ZEB1 (zinc finger E-box binding homeobox 1), and ZEB2 (zinc finger E-box binding homeobox 2) were compared across clear cell renal cell carcinoma cancer (ccRCC) patients (
Example 3. Transient Heterochromatin Inhibition Causes an Epigenetic Change, Stably Reversing EMT and Chemotherapy Resistance in Cancer Models
[0405] Cell surface markers can be used to identify mesenchymal, TIC cells. In breast cancer, the presence of CD44 and absence of CD24 markers are indicators of TICs. Specifically, cells expressing high levels of CD44 and no expression of CD24 are considered TICs, whereas cells expressing CD24 are considered non-TICs. HMLE cells display these cell surface markers upon overexpression of SNAIL or TWIST, which turns on the EMT transcriptional program and promotes a mesenchymal TIC state in cancer cells. To determine whether chaetocin treatment alters the mesenchymal phenotype of TICs, these markers were used to track the fate of HMLE-SNAIL cells following chaetocin treatment (1 μM) using fluorescence-activated cell sorting (FACS) analysis.
[0406] Epigenetic changes are those in which the phenotypic change induced upon transient exposure to a signal persists stably even in the absence of the inducing signal. In the discussion below, the ‘signal’ is treatment with chaetocin, which is hypothesized to convert mesenchymal cells into epithelial fate via inhibiting constitutive heterochromatin proteins, namely Suv39H1/H2. If heterochromatin proteins are central epigenetic regulators of EMT, a transient inhibition of heterochromatin activity should be sufficient to reverse EMT and, potentially, sensitize cells to chemotherapy. To determine whether a short pulse of the Suv39H1/H2 inhibitor, chaetocin, is sufficient to bring about an epigenetic change, cultured HMLE-TWIST cells (also referred to as ‘HMLE-TWIST TICs’ or ‘TICs’) were exposed to different doses of chaetocin for two days, followed by washout, and replacement with regular media. Cells were tracked for their phenotypic fate (e.g., CD44/CD24 marker expression) for another two or 6 days in chaetocin-free media. In this cell line, high CD44 and low CD24 expression indicate TIC cell populations. Cells that express CD24 are considered non-TICs. This waiting period was done because epigenetic changes often take a several days to take hold, often involving massive changes to the chromatin and transcriptional program of cells. The rationale for this experiment follows the logic that if chaetocin has caused an epigenetic change in TIC cells, reprogramming them out of the TIC phenotype, then a pulse of chaetocin should be sufficient to force cells out of the TIC phenotype, even in chaetocin free media. Stated in another way, a pulse of chaetocin may initiate a cellular program that ultimately forces cells to exit the TIC state.
[0407] To molecularly test whether chaetocin treatment converts mesenchymal cells into epithelial cells, HMLE-TWIST cells were treated with a pulse of chaetocin for 15 hours (15H), washed and then incubated in chaetocin-free media for 1 day (1D), 2 days (2D), and 4 days (4D). Levels of the ubiquitous epithelial marker E-cadherin (E-cad) was assayed using Western blots throughout the experiment.
Example 4. Transient Treatment with Chaetocin Sensitizes TIC-Rich Cancer Cells to Chemotherapy
[0408] To determine if EMT reversal with chaetocin can sensitize TIC-rich breast cancer cells, namely HMLE-TWIST cells, to conventional chemotherapeutic agents, cells were treated for 5 or 15 hours with different concentrations of chaetocin (20 nM, 50 nM, and 100 nM), as is schematized in
[0409] In a separate set of experiments, to test the generality of chaetocin in reversing the chemotherapy resistance of mesenchymal, TIC-rich cancers, well-characterized mesenchymal TIC-rich cell lines from ccRCC 786-0 and lung cancer cells (A549) were used along with HMLE-TWIST cells described above. As before, cells were treated with a pulse of chaetocin at low dose (80 nM) for 15 hours, after which the cells were washed and incubated for an additional 2 days in regular media. The cells were then treated with chemotherapeutic agents normally used to treat these cancers (cisplatin for renal and lung, and doxorubicin for breast cancer). Caspase-3 cleavage was used to measure apoptosis. As controls, cells were also treated with a single agent alone (inhibitor or chemotherapy), and experiments were performed in which the order of chaetocin and chemotherapy treatment were reversed. This was done to test a prediction of an epigenetic model: that the order of drug treatment is critical for reversing chemotherapy resistance.
Example 5. Identification of Specific Constitutive Heterochromatin Factors Whose Knockdown Converts Mesenchymal Cells into Epithelial Cells
[0410] If chaetocin epigenetic conversion of mesenchymal cells into epithelial cells is via inhibition of Suv39H1 and Suv39H2 proteins, then a knockdown of Suv39H1 and Suv39H2 should be sufficient to cause the same conversation. To test this hypothesis, Dicer-generated siRNA technology (dsiRNAs) was used to mediate the knockdown of central regulators of the heterochromatin pathway in HMLE-TWIST cells. Similar to before, the analysis was performed by tracking CD44/CD24 cell surface markers using FACS in HMLE-TWIST cells treated with different dsiRNA constructs.
[0411] The same experiments were subsequently performed using dsiRNAs targeting Suv39H2 (
Example 6. Cancer Cell Killing Efficacy of Chaetocin and its Derivatives
[0412] To determine the cancer cell killing efficacy of chaetocin and related molecules, cultured cancer cells were treated with either chaetocin, its monomeric analogue (Movassaghi Lab), or its dimeric analogue (Movassaghi Lab).
[0413] The monomeric analogue has the following structure:
##STR00017##
[0414] The dimeric analogue has the following structure:
##STR00018##
[0415] Two different cancer cell lines were tested, including chemotherapy-sensitive, human epithelial breast cancer cell lines (HMLE) (‘E’) and chemotherapy-resistant, TIC-rich, breast cancer cell line HMLE-TWIST (‘ET’). Of note, E and ET cells are genetically identical, wherein the only difference between them is overexpression of TWIST, one of the inducers of EMT, converting these cells into mesenchymal, TIC-rich chemotherapy resistant cells. Sensitivity and resistance to chemotherapy was defined on the basis of responsiveness of these cells to conventional chemotherapeutics. In
Example 7. Suv39H1 Expression is Higher in Mesenchymal, Tumor Initiating Cells Compared to Epithelial, Chemosensitive Cells
[0416] In this example, the levels of Suv39H1 protein in mesenchymal cells were investigated. Western blot analyses were performed on HMLE and HMLE-TWIST cells, and WT-7 and 786-O cells. Human mammary epithelial cells (HMLE) cells, overexpressing the human telomerase reverse transcriptase (hTERT) and simian virus 40 (SV40) T antigen is an epithelial cell lines that is especially sensitive to chemotherapeutic agents. On the other hand, HMLE-TWIST (E-TWIST) and HMLE-SNAIL (E-SNAIL) cell line are HMLE cell line in which TWIST (Snai2) or SNAIL (Snail) is overexpressed. SNAIL and TWIST are transcription factors whose overexpression converts epithelial, TIC-deplete, chemotherapy sensitive cancer cells into mesenchymal, TIC-rich, chemotherapy resistant cell lines through a process called epithelial-to-mesenchymal transition. Also, 786-O is a clear cell renal cell carcinoma (ccRCC) cell line, which is defective in the Von Hippel-Lindau tumor suppressor (VHL), enriched in TIC, and difficult to eliminate by conventional chemotherapy in patients carrying this mutation. WT7 cell line is a modified 786-O cell line in which VHL is re-introduced artificially, rendering them epithelial, TIC-depleted and susceptible to chemotherapy. These cell lines were used because they are otherwise genetically identical except for the presence of a single gene whose expression impacts EMT. Using an antibody against Suv39H1 (8792, Cell Signaling Technology), we found that Suv39H1 protein level is significantly higher in both mesenchymal cell lines (
[0417] The data in this Example together with the data presented in the rest of this application suggest that (1) Suv39H1 expression can be used as a marker for chemotherapy resistance, and (2) Suv39H1 is a target for eliminating EMT-induced resistance.
Example 8. Suv39H1 Physically Interacts with SNAIL in Mesenchymal, Tumor Initiating Cells
[0418] In this example, the mechanism of how Suv39H1 regulates and helps establish the mesenchymal state was investigated. To that end, the presence of any physical interactions between Suv39H1 and the EMT transcription factor, SNAIL, was assessed. Such an interaction would provide a molecular basis for how Suv39H1 is recruited to specific genes in mesenchymal cells, specifically those which are bound by SNAIL. Co-immunoprecipitation (Co-IP) experiments were performed using anti-Suv39H1 antibody. The results revealed that Suv39H1 physically interacts with Snail in HMLE-SNAIL and HMLE-TWIST cells (
[0419] Considering the observed Suv39H1-SNAIL interaction, inhibition of Suv39H1 can reverse EMT-induced chemotherapy resistance by transcriptional reprogramming of mesenchymal cells. Because Suv39H1 is involved in repression of gene expression, its inhibition can lead to activation of SNAIL-repressed genes.
Example 9. Chaetocin Treatment Causes an Increase in Expression of Epithelial and a Decrease in Expression of Mesenchymal Genes
[0420] The data presented herein demonstrate that a short treatment with chaetocin alters the mesenchymal properties of cells as assayed by CD44 and CD24 marks on HMLE-TWIST cell lines. To determine whether the transcriptional changes associated with chaetocin treatment support this apparent change in cell state (reversal of mesenchymal fate), RNA-seq experiments were performed on cells treated for 15 hours with 0.3 μM of chaetocin after which total RNA was extracted and subjected to deep sequencing.
TABLE-US-00004 TABLE 4 Epithelial Genes Whose Expression Is Increased in Response to Chaetocin Treatment Adjusted Gene name Log2foldChange P value E-cadherin CDH1 4.25 0.00130 Collagen 2(A2) COL2A1 4.34 7.06E−05 survivin-1 BIRC5 0.491 0.000580 Keratins KRT8 1.30 2.05E−24 KRT18 1.95 2.87E−42 KRT34 5.49 1.66E−16 KRT7 1.59 0.000232 KRT80 1.24 9.08E−06 KRT19 4.46 0.000532 KRT81 1.47 0.00120 KRT86 3.10 1.91E−06 Claudins CLDN6 7.30 4.56E−19 CLDN4 3.68 6.97E−12 CLDN2 6.00 0.000950 CLDN9 4.59 0.000257 CLDN23 1.18 0.0296 CLDN16 2.77 0.0564 increased expression in epithelial cells CRB1 9.40 6.37E−09 CST6 2.12 0.0520
TABLE-US-00005 TABLE 5 Mesenchymal Genes and Cancer Stem Cell Markers Adjusted Gene name Log2foldChange P value Mesenchymal genes whose expression is repressed in response to chaetocin treatment EMT TFs TWIST1 −0.549 7.04E−05 SNAI2 −0.623 0.000479 ZEB1 −0.666 9.06E−07 Activated by TWIST LAMB1 −1.05 5.47E−15 Activated by Slug MMP14 −1.14 7.43E−13 Activated in WISP1 −1.06 1.81E−07 mesenchymal cells Cancer Stem cell markers whose expression decrease in response to chaetocin treatment CD24 −0.912 GLI-2 −0.941 ALDH1A1 −2.01 Errb2 −0.829 GJA1 −0.627 PTEN −0.780 Nanog −2.21 O ct4 −1.76 NRF-2 −0.862
Example 10. Chaetocin Treatment Causes a Decrease in H3K9Me3 in Mesenchymal Cells
[0421] Chaetocin is an inhibitor of Suv39H1 and Suv39H2 enzymes, whose catalytic activities trimethylates lysine 9 of histone H3. To demonstrate that a short pulse of chaetocin can result in a decrease in H3K9me3, we performed chromatin immunoprecipitation experiments using a specific H3K9me3 antibody (abcam) followed by deep sequencing of the purified DNA fragment.
TABLE-US-00006 TABLE 6 Gene Ontology Biological Process (BPs) Enriched among Genes Which Lose H3K9me3 Upon Chaetocin Treatment (0.3 uM for 15 hours) Fold # # expected Enrichment +/− raw P value FDR cell-cell adhesion 494 27 10.82 2.49 + 0.0000358 0.0227 neuron differentiation 992 46 21.73 2.12 + 0.00000428 0.00452
TABLE-US-00007 TABLE 7 Genes Which Display a Loss of H3K9me and Transcriptional Repression in Response to Chaetocin Treatment (see FIGS. 10 and 11) PRKCZ FAM20C GABBR2 DNAH10 ZFP3 ZNF556 EPHA8 CARD11 CFL1 TMEM132B LRRC46 DOHH SAG PPP1R9A DLG2 EEF1AKMT1 USP36 VAV1 ITGA9 PTPRN2 NTM C13orf46 LDLRAD4 ZNF557 NEK11 PNPLA4 CTNNA3 HSP90AA1 NFATC1 ZNF333 TACC3 PPP1R3F LRMDA ATP8B4 CTDP1 CPAMD8 FRAS1 CCNB3 JAKMIP3 RASGRF1 ZNF341 RYR1 ZFP2 MYOM2 CD163L1 HYDIN COL20A1 SPTBN4 GRM4 PXDNL KRT86 OSGIN1 MADCAM1 HSF2BP DNAH8 C8orf34 KRT81 TUBB3 HCN2 ITGB2
[0422] The genes presented in Table 7 are overrepresented in heterotypic cell-cell adhesion and receptor clustering gene ontology biological processes, among which the heterotypic cell-cell adhesion gene ontology is consistent with increase in epithelialization of mesenchymal HMLE-TWIST cells.
[0423] Suv39H1 inhibition can reverse EMT-induced chemotherapy resistance by transcriptional reprogramming of mesenchymal cells. Considering the role of Suv39H1 in repressing transcription, inhibition of Suv39H1 can lead to activation of SNAIL-repressed genes.
Example 11. Chaetocin Treatment Reprograms Mesenchymal Cells into an Epithelial Fate
[0424] Using FACS (see
TABLE-US-00008 TABLE 8 Adjusted Term P-value P-value Genes Pathways down-regulated in HMLE-TWIST cells after two days of treatment with 150 μM of chaetocin miRNA targets in ECM and 1.95E−11 9.20E−09 LAMB2; LAMA4; ITGA1; FN1; membrane receptors WP2911 LAMC1; THBS1; COL3A1; COL1A2; COL5A1; COL4A1; COL5A3; COL5A2; ITGA11 Focal Adhesion-PI3K-Akt-mTOR- 1.20E−09 2.82E−07 LAMA5; CSF1; ATF6B; EPAS1; signaling pathway WP3932 IRS1; ITGB4; LAMA4; ITGA2B; PTEN; PIK3CD; IRS2; LAMC1; THBS1; FOXO1; PIK3C2B; AKT2; PDGFC; ITGAV; IL6R; MAPK3; PDGFRB; SREBF1; PDGFRA; ANGPT1; LAMB2; FN1; PPP2R5A; LAMB1; EPOR; COL1A1; COL3A1; KITLG; COL1A2; COL5A1; COL4A1; COL4A4; ITGA10; COL5A3; COL5A2; ITGA11; ULK1; ITGA5; FGF13; FGFR3; ATF4; FGFR1 Focal Adhesion WP306 9.06E−08 1.42E−05 LAMA5; ITGB4; LAMA4; ITGA2B; PXN; PTEN; PIK3CD; LAMC1; THBS1; CCND3; AKT2; PDGFC; ERBB2; RAC3; ITGAV; MAPK3; PDGFRB; PDGFRA; CAV2; LAMB2; ITGA1; FN1; LAMB1; COL1A1; COL1A2; COL4A1; COL4A4; ITGA10; COL5A3; COL5A2; ITGA11; ITGA5 Hypothesized Pathways in 5.52E−06 6.51E−04 FBN2; TGFBR3; SERPINE1; Pathogenesis of Cardiovascular LTBP1; RUNX2; CTGF; MAPK3; Disease WP3668 TGFBR2; FBN1 PI3K-Akt Signaling Pathway 1.17E−05 0.00110303 LAMA5; CSF1; ATF6B; IRS1; WP4172 ITGB4; LAMA4; ITGA2B; PTEN; PIK3CD; LAMC1; THBS1; CCND3; AKT2; PDGFC; ITGAV; IL6R; MAPK3; PDGFRB; PDGFRA; ANGPT1; LAMB2; ITGA1; FN1; PPP2R5A; LAMB1; EPOR; COL1A1; KITLG; COL1A2; COL4A1; COL4A4; ITGA10; ITGA11; COL4A5; PKN1; ITGA5; FGF13; FGFR3; ATF4; FGFR1 Inflammatory Response Pathway 2.98E−05 0.00234525 COL1A1; LAMA5; COL3A1; WP453 COL1A2; LAMB2; FN1; LAMB1; LAMC1; THBS1 miR-509-3p alteration of 2.44E−04 0.01646427 COL1A1; EDNRA; COL3A1; YAP1/ECM axis WP3967 SPARC; COL5A1; FN1 Alpha 6 Beta 4 signaling pathway 4.23E−04 0.02492966 LAMA5; IRS1; ITGB4; LAMB2; WP244 IRS2; LAMB1; LAMC1; MAPK3 Integrin-mediated Cell Adhesion 5.91E−04 0.03100336 CAV2; ITGB4; ITGA2B; PXN; WP185 ITGA1; ARAF; CAPN6; AKT2; ITGA10; ITGA11; RAC3; ITGAV; CAPN1; ITGA5; TNS1 Primary Focal Segmental 7.19E−04 0.033914 SCARB2; LAMA5; INF2; CD151; Glomerulosclerosis FSGS ITGB4; LAMB2; COL4A4; COL4A5; WP2572 ITGAV; AGRN; DKK1; DNM1 Epithelial to mesenchymal 7.40E−04 0.03175257 NOTCH2; FZD1; NOTCH3; transition in colorectal cancer SPARC; JUP; MMP2; WNT5A; WP4239 FZD6; FN1; PIK3CD; TGFBR2; WNT6; LATS2; WNT11; COL4A1; AKT2; COL4A4; COL4A5; ITGA5; MAPK3 Oncostatin M Signaling Pathway 0.00103803 0.04082935 PIAS3; EGR1; IRS1; SERPINE1; WP2374 PXN; PTK2B; RICTOR; IL6ST; LDLR; CYR61; MAPK3 Adipogenesis WP236 0.00116354 0.04224551 FZD1; NCOA1; SREBF1; CEBPD; IRS1; EPAS1; STAT2; SERPINE1; IRS2; FOXO1; KLF6; BMP1; SPOCK1; IL6ST; PPARA; LPIN1; LPIN3 Hippo-Merlin Signaling 0.00130362 0.04395064 PDGFRB; PDGFRA; ITGB4; Dysregulation WP4541 ITGA2B; ITGA1; YY1AP1; CTGF; LATS2; PLCB4; ITGA10; ITGA11; ITGAV; CDH15; ITGA5; FGFR3; FGFR1 SREBF and miR33 in cholesterol 0.00154147 0.04850487 ABCA1; SREBF1; PPARA; MED15; and lipid homeostasis WP2011 LDLR Pathways down-regulated in HMLE-TWIST cells after two days of treatment with 500 μM of chaetocin miRNA targets in ECM and 2.85E−09 1.35E−06 LAMB2; LAMA4; SDC2; ITGA1; membrane receptors WP2911 LAMC1; THBS1; COL3A1; COL1A2; COL5A1; COL4A2; COL4A1; COL5A2; ITGA11 Epithelial to mesenchymal 1.30E−06 3.07E−04 NOTCH3; SPARC; LRP5; TWIST1; transition in colorectal cancer PIK3CD; HIF1A; PKD1; LRP6; WP4239 WNT6; WNT11; AKT2; MEF2D; MAPK3; FZD1; TGFB2; JUP; MMP2; WNT5A; FZD6; MAPK14; MPP5; TGFBR2; LATS2; COL4A2; PIK3CA; COL4A1; MMP15; COL4A4; TRAF6; COL4A3; COL4A6; COL4A5; ITGA5 Focal Adhesion WP306 1.65E−06 2.59E−04 ITGB1; LAMA5; ROCK1; LAMA4; ITGA2B; PXN; PTEN; PIK3CD; LAMC1; THBS1; PPP1CB; PPP1CC; CCND3; AKT2; PDGFC; ERBB2; RAC3; ITGAV; PAK4; MAPK3; PDGFRB; PDGFRA; ITGA4; CAV2; LAMB2; ITGA1; LAMB1; COL1A2; COL4A2; PIK3CA; COL4A1; COL4A4; ITGA10; COL5A2; ITGA11; RAPGEF1; COL4A6; ITGA5 Focal Adhesion-PI3K-Akt-mTOR- 9.69E−06 0.00114365 ITGB1; ATF2; CDKN1A; CSF1; signaling pathway WP3932 IRS1; ITGA2B; PTEN; PIK3CD; IRS2; LAMC1; PIK3C2B; AKT2; ITGAV; PDGFRB; PDGFRA; ITGA4; STRADA; F2R; TSC2; COL4A2; PIK3CA; COL4A1; COL4A4; COL4A6; ULK1; ITGA5; ATF4; LAMA5; ATF6B; LAMA4; LPAR1; HIF1A; THBS1; FOXO1; HSP90B1; PDGFC; MAPK3; ANGPT1; LAMB2; LAMB1; COL3A1; KITLG; COL1A2; COL5A1; ITGA10; COL5A2; ITGA11; RPS6KB2; FGFR1 Oncostatin M Signaling Pathway 2.17E−05 0.00204591 PIAS3; IRS1; SERPINE1; PXN; WP2374 TYK2; MAPK14; HIF1A; CYR61; NFKBIA; CASP7; CDK2; PTK2B; CCL2; RICTOR; IL6ST; LDLR; MAPK3 Primary Focal Segmental 2.44E−05 0.00192295 SCARB2; ITGB1; LAMA5; Glomerulosclerosis FSGS CDKN1A; CD151; LAMB2; LRP5; WP2572 DKK1; DNM1; CD2AP; LRP6; INF2; COL4A4; COL4A3; COL4A5; ITGAV; VIM; AGRN Angiopoietin Like Protein 8 1.17E−04 0.00790986 THRA; IRS1; PIK3CD; CBLB; Regulatory Pathway WP3915 IRS2; FOXO1; AKT2; RICTOR; SLC16A2; MAP2K7; MAP4K3; MAPK3; MAP4K2; PRKAB2; MAP3K1; MINK1; TSC2; MAPK14; SREBF2; PIK3CA; RPS6KB2; RAPGEF1; TRIP10; RHOQ; MAP3K12 Hypothesized Pathways in 1.34E−04 0.00791924 FBN2; POSTN; SERPINE1; Pathogenesis of Cardiovascular MAPK14; LTBP1; CTGF; MAPK3; Disease WP3668 TGFBR2; FBN1 Adipogenesis WP236 2.38E−04 0.01250733 FZD1; NCOA1; NCOA2; CDKN1A; CEBPD; IRS1; STAT2; SERPINE1; TWIST1; IRS2; HIF1A; FOXO1; NCOR2; RBL2; KLF6; NCOR1; ID3; SPOCK1; FAS; IL6ST; PPARA; MEF2D; LPIN3; PPARD Signaling Pathways in 4.69E−04 0.02214927 PDGFRB; PDGFRA; CDKN1A; Glioblastoma WP2261 IRS1; PTEN; PIK3CD; TSC2; FOXO4; FOXO1; PIK3C2B; PIK3CA; AKT2; ERBB2; CDK2; MAP2K7; MAPK3; FGFR1 IL-1 signaling pathway WP195 5.77E−04 0.02476774 ATF2; MAP3K1; IL1R1; MAPK14; NFKBIA; TRAF6; MAPKAPK2; CCL2; TAB3; TAB2; UBE2V1; MAP2K7; MAPK3 Structural Pathway of Interleukin 1 6.61E−04 0.02601238 NFKBIA; ATF2; MAP3K1; IL1R1; (IL-1) WP2637 TRAF6; MAPKAPK2; IRF7; TAB3; TAB2; MAP2K7; MAPK14; MAPK3 PI3K-Akt Signaling Pathway 6.62E−04 0.02403748 ITGB1; ATF2; LAMA5; CDKN1A; WP4172 CSF1; ATF6B; IRS1; LAMA4; ITGA2B; LPAR1; PTEN; PIK3CD; LAMC1; THBS1; HSP90B1; CCND3; AKT2; PDGFC; ITGAV; MAPK3; PDGFRB; PDGFRA; ITGA4; ANGPT1; LAMB2; F2R; ITGA1; TSC2; LAMB1; RBL2; KITLG; COL1A2; COL4A2; PIK3CA; COL4A1; COL4A4; ITGA10; COL4A3; ITGA11; CDK2; RPS6KB2; COL4A6; COL4A5; PKN1; ITGA5; ATF4; FGFR1 Integrin-mediated Cell Adhesion 8.04E−04 0.02710242 ITGB1; ITGA4; ROCK1; CAV2; WP185 ITGA2B; PXN; ITGA1; CAPN6; CAPN7; AKT2; ITGA10; ITGA11; RAPGEF1; RAC3; ITGAV; CAPN1; ITGA5; TNS1; PAK4 Insulin Signaling WP481 0.00103294 0.03250332 STXBP3; IRS1; INPPL1; PTEN; PIK3CD; CBLB; IRS2; FOXO1; AKT2; MAP2K7; MAP4K3; MAPK3; MAP4K2; MAP3K1; MINK1; TSC2; MAPK14; ARHGAP33; EHD2; PFKL; PIK3CA; RPS6KB2; RAPGEF1; VAMP2; RHOQ; MAP3K12 Wnt/beta-catenin Signaling 0.00105563 0.0311412 JUP; ZBTB16; FZD6; LRP5; DKK1; Pathway in Leukemia WP3658 PML; LRP6; PPARD RIG-I-like Receptor Signaling 0.00137809 0.03826237 DDX17; OTUD5; MAP3K1; WP3865 MAPK14; ATG12; NFKBIA; CYLD; MAVS; TRAF6; IRF7; PIN1; TBKBP1; AZI2 MicroRNAs in cardiomyocyte 0.00175991 0.04614883 FZD1; HDAC5; CAMK2D; ROCK1; hypertrophy WP1544 WNT5A; LRP5; PIK3CD; MAPK14; LRP6; HDAC7; NFATC4; PIK3CA; AKT2; IL6ST; MAP2K7; MAPK3 Sterol Regulatory Element- 0.00179121 0.04449747 PRKAB2; SEC23A; SEC24A; Binding Proteins (SREBP) INSIG1; MED15; SREBF2; signalling WP1982 PIK3CA; MVD; ACSS1; SEC24D; ATF6; LDLR; SEC24C; SEC31A NOTCH1 regulation of human 0.00205955 0.04860529 ITGA1; MGP; LPAR1; CALU; endothelial cell calcification PLAT; SAT1 WP3413 miR-509-3p alteration of 0.00205955 0.04629075 EDNRA; COL3A1; SPARC; YAP1/ECM axis WP3967 COL5A1; TWIST1; GPC6 Pathways up-regulated in HMLE-TWIST cells after two days of treatment with 150 μM of chaetocin *DNA IR-damage and cellular 6.01E−08 2.84E−05 FEN1; PCNA; RMI1; XRCC5; response via ATR WP4016 H2AFX; FANCA; BRCA1; BRCC3; BRCA2; MLH1; BRIP1; RAD50; CDC45; TRIM28; EXO1; CHEK2; E2F1; USP1; RBBP8; CLSPN; TP53; ATRIP; ATR Histone Modifications WP2369 1.06E−06 2.49E−04 SETD4; HIST1H3J; SETD9; HIST1H4L; SETD1B; SETMAR; HIST1H4A; HIST1H3A; HIST1H3F; HIST1H3G; HIST1H4H; HIST1H3H; HIST1H4I; HIST2H3D; HIST1H3B; HIST1H4C; HIST1H3C; HIST1H3D; HIST1H4F The effect of progerin on the 3.85E−06 6.05E−04 MBD3; HIST1H3J; HIST1H3A; involved genes in Hutchinson- E2F1; HIST1H3F; INPP5K; Gilford Progeria Syndrome HIST1H3G; HIST1H3H; WP4320 HIST1H3B; HIST2H3D; HIST1H3C; TP53; HIST1H3D *Retinoblastoma Gene in Cancer 1.85E−05 0.00217712 CDKN1B; RFC4; PCNA; RRM2; WP2446 CDC7; HMGB1; CDC25A; SMC2; SAP30; DHFR; POLA1; CDC45; CCNE2; ORC1; KIF4A; HLTF; MCM3; E2F1; E2F2; TP53 DNA IR-Double Strand Breaks 2.38E−05 0.0022439 BLM; DCLRE1C; PCNA; ACTL6A; (DSBs) and cellular response via H2AFX; BRCA1; BRCA2; RAD50; ATM WP3959 TRIM28; CDK5; EXO1; CHEK2; E2F1; TP53; ATR DNA Damage Response WP707 2.42E−05 0.00190659 CDKN1B; H2AFX; BRCA1; CDC25A; CCNB3; RAD50; CASP8; RRM2B; CCNE2; CDK5; CHEK2; E2F1; CYCS; SFN; TP53; ATRIP; ATR miRNA Regulation of DNA 4.41E−05 0.00297619 CDKN1B; H2AFX; BRCA1; Damage Response WP1530 CDC25A; CCNB3; RAD50; CASP8; RRM2B; CCNE2; CDK5; CHEK2; E2F1; CYCS; SFN; TP53; ATRIP; ATR NRF2-ARE regulation WP4357 4.55E−05 0.002686 NQO1; AIMP2; GCLC; YES1; KEAP1; HMOX1; PGAM5; SLC7A11; GCLM Phytochemical activity on NRF2 8.65E−05 0.0045345 NQO1; AIMP2; GCLC; KEAP1; transcriptional activation WP3 HMOX1; SLC7A11; GCLM DNA Replication WP466 9.38E−05 0.00442817 ORC5; POLA1; PCNA; ORC6; CDC45; RFC4; ORC1; UBC; MCM3; MCM10; CDC7; CDC6 Cell Cycle WP179 2.68E−04 0.01148935 CDKN2D; CDKN1B; PCNA; YWHAB; CDC7; CDC6; PKMYT1; CDC25A; CCNB3; ORC5; ORC6; CDC45; CCNE2; ORC1; ESPL1; CHEK2; MCM3; E2F1; E2F2; SFN; TP53; ATR G1 to S cell cycle control WP45 5.53E−04 0.02173412 CDKN2D; CDKN1B; PCNA; CDC25A; ORC5; ORC6; CDC45; CCNE2; ORC1; CREB3L4; MCM3; E2F1; E2F2; TP53 *ATM Signaling Network in 7.97E−04 0.02892879 G6PD; RAD50; DCLRE1C; Development and Disease TRIM28; CDK5; CHEK2; WP3878 RASGRF1; H2AFX; RBBP8; AURKB; ATR Pyrimidine metabolism WP4022 0.00131202 0.04423398 DTYMK; DUT; PNPT1; RRM2; CTPS1; NME1; DCTD; POLA1; RRM2B; POLR2A; POLR3B; POLR3G; NT5M; DCTPP1; POLR3K; POLR2L Pathways up-regulated in HMLE-TWIST cells after two days of treatment with 500 μM of chaetocin *DNA IR-damage and cellular 3.54E−06 0.00167164 MDC1; PCNA; POLN; H2AFX; response via ATR WP4016 BRCA1; BRCC3; BRCA2; MLH1; PALB2; BRIP1; RAD50; CDC45; RAD51; MSH2; EXO1; FANCD2; USP1; RBBP8; CLSPN; TP53; ATRIP; MCM2; ATR DNA IR-Double Strand Breaks 5.41E−06 0.00127716 MDC1; BLM; DCLRE1C; PCNA; (DSBs) and cellular response via ACTL6A; H2AFX; BRCA1; BRCA2; ATM WP3959 TERF2; RAD50; RAD51; CDK5; EXO1; FANCD2; CASP3; NABP2; TP53; ATR miRNA Regulation of DNA 2.10E−05 0.00330261 MCM7; GADD45A; H2AFX; Damage Response WP1530 BRCA1; CDC25A; CDC20B; CCNB3; CCNB1; RAD50; RAD51; CCND1; CCNE2; CDK5; FANCD2; CASP3; CYCS; SFN; TP53; ATRIP; ATR Cell Cycle WP179 2.66E−05 0.00314451 PCNA; MCM7; YWHAB; PKMYT1; CDC20; CCNB3; ORC5; CCNB1; ORC6; CDC45; CCND1; E2F2; SFN; E2F3; E2F4; YWHAH; GADD45A; CDC7; CDC6; CDC25A; STAG1; DBF4; CCNE2; MCM3; TP53; MAD1L1; ATR; MCM2 DNA Damage Response WP707 1.30E−04 0.01225552 GADD45A; H2AFX; BRCA1; CDC25A; CCNB3; CCNB1; RAD50; RAD51; CCND1; CCNE2; CDK5; FANCD2; CASP3; CYCS; SFN; TP53; ATRIP; ATR DNA Replication WP466 2.04E−04 0.0160872 PCNA; RFC4; MCM7; MCM10; CDC7; CDC6; ORC5; ORC6; CDC45; DBF4; UBC; MCM3; MCM2 *Retinoblastoma Gene in Cancer 4.43E−04 0.02986511 DNMT1; RFC4; PCNA: RRM2; WP2446 MCM7; CDC7; HMGB1; CDC25A; SAP30; DHFR; CCNB1; CDC45; CCND1; CCNE2; KIF4A; STMN1; MCM3; E2F2; E2F3; TP53 G1 to S cell cycle control WP45 6.05E−04 0.0357193 PCNA; MCM7; GADD45A; CDC25A; ORC5; CCNB1; ORC6; CDC45; CCND1; CCNE2; CREB3L4; MCM3; E2F2; E2F3; TP53; MCM2
TABLE-US-00009 TABLE 9 Adjusted Term P-value P-value Genes Pathways down-regulated in HMLE-TWIST cells two days after chaetocin removal (@150 μM) miRNA targets in ECM and 2.16E−09 1.02E−06 COL3A1; COL1A2; COL5A1; membrane receptors WP2911 COL4A2; LAMB2; COL4A1; LAMA4; SDC2; ITGA1; COL5A2; FN1 Focal Adhesion WP306 2.52E−06 5.94E−04 FLT1; SHC1; LAMA4; ITGA2B; AKT2; PDGFC; ERBB2; RAC3; ITGAV; PAK4; MAPK3; PDGFRB; PDGFRA; ITGA4; CAV2; LAMB2; ITGA1; FN1; LAMB1; COL1A1; COL1A2; COL4A2; COL4A1; COL4A4; ITGA10; COL5A2; COL4A6 Focal Adhesion-PI3K-Akt-mTOR- 1.14E−05 0.00178759 FLT1; CSF1; ATF6B; IRS1; signaling pathway WP3932 LAMA4; ITGA2B; LPAR1; IRS2; GYS1; AKT2; PDGFC; ITGAV; MAPK3; PDGFRB; SREBF1; PDGFRA; ITGA4; ANGPT1; LAMB2; FN1; LAMB1; EPOR; COL1A1; EFNA1; COL3A1; COL1A2; COL4A2; COL5A1; COL4A1; COL4A4; ITGA10; GNB2; COL5A2; COL4A6 PI3K-Akt Signaling Pathway 5.28E−05 0.00622965 FLT1; CSF1; ATF6B; PKN3; WP4172 IRS1; LAMA4; ITGA2B; LPAR1; GYS1; AKT2; PDGFC; ITGAV; MAPK3; PDGFRB; PDGFRA; ITGA4; ANGPT1; LAMB2; ITGA1; FN1; LAMB1; EPOR; COL1A1; EFNA1; COL1A2; COL4A2; COL4A1; COL4A4; ITGA10; GNB2; CDK2; COL4A6; COL4A5; PKN1; SOS2 Inflammatory Response Pathway 8.84E−05 0.00834457 COL1A1; CD40; COL3A1; WP453 COL1A2; LAMB2; FN1; LAMB1; TNFRSF1A miR-509-3p alteration of 1.24E−04 0.00978137 COL1A1; COL3A1; SPARC; YAP1/ECM axis WP3967 COL5A1; FN1; GPC6 Complement and Coagulation 1.31E−04 0.00883403 C3; SERPINA1; C6; PROC; Cascades WP558 C1S; C7; C1R; PROS1; SERPING1; TFPI; SERPINA5 Epithelial to mesenchymal 1.52E−04 0.00899473 NOTCH3; TGFB2; SPARC; transition in colorectal cancer SHC1; MMP2; WNT5A; FZD6; WP4239 FN1; LRP5; PKD1; LATS2; COL4A2; COL4A1; CTDSP1; AKT2; COL4A4; COL4A6; COL4A5; SOS2; MAPK3 Hypothesized Pathways in 1.76E−04 0.00921742 FBN2; TGFBR3; POSTN; SHC1; Pathogenesis of Cardiovascular LTBP1; MAPK3; FBN1 Disease WP3668 Human Complement System 3.54E−04 0.01671749 CD40; C1S; C1R; PROS1; WP2806 ITGA2B; LAMB1; DCN; GNAI2; C3; C6; C7; SERPING1; CALR; PRKACA Pathways down-regulated in HMLE-TWIST cells two days after chaetocin removal (@500 μM) Cytoplasmic Ribosomal Proteins 2.45E−18 1.16E−15 RPL4; RPL30; RPL3; RPL32; WP477 RPL10; RPL31; RPL34; RPL12; RPLP0; RPL11; RPL10A; RPL9; RPS4X; RPS6KA3; RPL7A; RPS15A; RPS18; RPL37; RPS2; RPS27A; RPL39; RPS13; RPS12; RPS9; RPL41; RPL21; RPS8; RPL22; RPS6; RPL13A; RPL35A; RPS3A; RPSA; RPL23A; RPS27; RPS29; RPS6KB2; RPL24; RPL29 miRNA targets in ECM and 3.09E−10 7.30E−08 LAMB2; LAMA4; SDC2; ITGA1; membrane receptors WP2911 FN1; LAMC1; THBS1; COL3A1; COL1A2; COL5A1; COL4A2; COL4A1; COL5A2; COL6A3 Focal Adhesion WP306 3.50E−09 5.50E−07 ITGB1; ROCK1; SHC1; LAMA4; PTEN; ILK; LAMC1; ARHGAP5; THBS1; PPP1CB; PPP1CC; CCND3; AKT3; PDGFC; ERBB2; RAC3; MAPK1; ITGAV; PAK4; MAPK3; PDGFRB; PDGFRA; PPP1R12A; ITGA4; CAV2; LAMB2; CAV1; ITGA1; FN1; PARVA; LAMB1; ACTN4; COL1A1; COL1A2; COL4A2; PIK3CA; COL4A1; COL4A4; ITGA10; COL5A2; COL4A6; CTNNB1; DOCK1; VCL; BIRC2 Primary Focal Segmental 7.22E−07 8.52E−05 SCARB2; ITGB1; CD151; MME; Glomerulosclerosis FSGS LAMB2; ILK; PARVA; ACTN4; WP2572 DKK1; CD2AP; LRP6; INF2; COL4A4; COL4A5; CTNNB1; ITGAV; VIM; UTRN; TLR4; VCL; NCK1 Focal Adhesion-PI3K-Akt-mTOR- 1.50E−06 1.41E−04 ITGB1; ATF2; CSF1; IRS1; signaling pathway WP3932 PTEN; SLC2A3; LAMC1; PIK3C2B; FGF7; CREB3L2; AKT3; ITGAV; JAK1; PDGFRB; PDGFRA; ITGA4; RPS6; F2R; PPP2R5D; TBC1D1; COL4A2; PIK3CA; COL4A1; COL4A4; COL4A6; ATF6B; LAMA4; LPAR1; HIF1A; THBS1; HSP90B1; NRAS; GNG2; PDGFC; MAPK1; EIF4B; MAPK3; ANGPT1; LAMB2; FN1; LAMB1; COL1A1; EFNA1; COL3A1; KITLG; COL1A2; COL5A1; ITGA10; GNB2; COL5A2; RPS6KB2; GNB4; KRAS EGF/EGFR Signaling Pathway 1.95E−06 1.53E−04 ROCK1; SHC1; INPPL1; PTEN; WP437 PEBP1; CBLB; AP2A1; IQGAP1; PLD1; PLD2; PIK3C2B; RPS6KA3; GJA1; ERBB2; REPS2; MAPK1; RICTOR; EPS15; AP2M1; NCK1; JAK1; STAT5B; PRKCI; MAP3K1; CAV2; CAV1; AP2B1; MAPK14; PLSCR1; SP1; RASA1; PXDN; KRAS; STAM2 Nonalcoholic fatty liver disease 5.56E−06 3.75E−04 COX7B; NDUFB7; UQCRB; WP4396 IRS1; NDUFA12; NDUFB11; COX7A2; UQCR10; COX5B; COX7C; UQCRH; CASP7; AKT3; LEPR; CCL2; CYC1; NDUFV1; PRKAB2; NDUFA5; NDUFA4; NDUFA2; NDUFA1; SDHD; COX6B1; TNFRSF1A; PIK3CA; NDUFS5; UQCRC1; NDUFS3; NDUFS2; NDUFS1; UQCRC2 Ebola Virus Pathway on Host 7.78E−06 4.59E−04 ITGB1; CLTC; IQGAP1; MAPK1; WP4217 ITGAV; EPS15; CTSB; MAPK3; GSN; ITGA4; CAV2; CAV1; ITGA1; HLA-B; HLA-C; EIF2AK2; ACTN4; HLA-A; HLA- F; RHOC; HLA-E; RHOB; PIK3CA; NPC2; TYRO3; RAB9A; MFGE8; TLR4 Inhibition of exosome biogenesis 8.09E−06 4.24E−04 RAB5B; NRAS; PDCD6IP; and secretion by Manumycin A in RRAS; ARAF; RAB27A; MAPK1; CRPC cells WP4301 KRAS; MAPK3 PI3K-Akt Signaling Pathway 1.08E−05 5.11E−04 ITGB1; ATF2; CSF1; IRS1; WP4172 PTEN; LAMC1; CCND3; FGF7; CREB3L2; AKT3; ITGAV; JAK1; PDGFRB; PDGFRA; ITGA4; RPS6; F2R; ITGA1; PPP2R5D; RBL2; COL4A2; PIK3CA; COL4A1; COL4A4; COL4A6; COL4A5; COL6A3; TLR4; ATF6B; LAMA4; LPAR1; THBS1; HSP90B1; NRAS; GNG2; PDGFC; MAPK1; EIF4B; MAPK3; ANGPT1; LAMB2; FN1; LAMB1; COL1A1; EFNA1; KITLG; COL1A2; G6PC3; ITGA10; GNB2; CDK2; RPS6KB2; GNB4; KRAS; PKN1 Sterol Regulatory Element- 2.42E−05 0.00104014 FDPS; SEC13; PRKAB2; Binding Proteins (SREBP) SEC23A; SAR1A; INSIG1; signalling WP1982 CYP51A1; DBI; SREBF2; SQLE; PIK3CA; SP1; SEC24D; ATF6; LDLR; SEC24C; KPNB1; SEC31A Electron Transport Chain 3.04E−05 0.00119557 SURF1; COX7B; NDUFB7; (OXPHOS system in UQCRB; NDUFA5; NDUFA12; mitochondria) WP111 NDUFA4; NDUFA2; NDUFA1; COX7A2; SDHD; UQCR10; COX5B; COX7C; UQCRH; COX6B1; NDUFS5; UQCRC1; NDUFS3; NDUFS2; NDUFS1; UQCRC2; NDUFV1 Integrin-mediated Cell Adhesion 6.69E−05 0.00242755 ITGB1; ITGA4; ROCK1; SHC1; WP185 CAV2; CAV1; ITGA1; ARAF; ILK; CAPNS1; CAPN6; CAPN7; ITGA10; AKT3; RAC3; MAPK1; ITGAV; CAPN1; DOCK1; VCL; TNS1; PAK4 Non-genomic actions of 1,25 1.29E−04 0.00436502 IFI27L2; CAV1; STAT2; dihydroxyvitamin D3 WP4341 MAPK14; IFI44L; TNFRSF1A; RXRB; NRAS; SP1; CCL2; MAPK1; KRAS; PLCB1; TLR4; CAMK2G; JAK1; MAPK3 Regulation of Actin Cytoskeleton 1.36E−04 0.00427095 ROCK1; BRK1; IQGAP1; WP51 PIK3C2B; FGF7; NRAS; RRAS; CFL2; TMSB4X; RAC3; MAPK1; PIP4K2B; MYH10; WASF2; PAK4; MAPK3; PDGFRB; PDGFRA; PPP1R12A; GSN; F2R; ITGA1; FN1; PIK3CA; KRAS; DOCK1; VCL; ARHGEF6 Oncostatin M Signaling Pathway 1.43E−04 0.00422021 STAT5B; IRS1; SHC1; RPS6; WP2374 MAPK14; HIF1A; CYR61; CASP7; CDK2; CCL2; MAPK1; RICTOR; KRAS; LDLR; JAK1; MAPK3 Inflammatory Response Pathway 1.76E−04 0.00489378 COL1A1; COL3A1; COL1A2; WP453 LAMB2; FN1; LAMB1; LAMC1; TNFRSF1B; THBS1; TNFRSF1A Hypothesized Pathways in 1.91E−04 0.00500609 FBN2; TGFBR3; POSTN; SHC1; Pathogenesis of Cardiovascular MAPK1; MAPK14; LTBP1; Disease WP3668 MAPK3; FBN1 DNA Damage Response (only 2.49E−04 0.00618968 MAP3K1; IRS1; SHC1; WNT5A; ATM dependent) WP710 PTEN; SOD2; PIK3C2B; RBL2; CCND3; NRAS; SCP2; BCL6; PIK3CA; AKT3; CAT; ERBB2; RAC3; MAPK1; CTNNB1; ATM; KRAS; LDLR RAC1/PAK1/p38/MMP2 Pathway 2.51E−04 0.00592705 ITGB1; STAT5B; ANGPT1; WP3303 MMP2; FN1; MAPK14; CASP7; NRAS; PIK3CA; RASA1; ERBB2; MAPK1; CTNNB1; KRAS; NCK1; MAPK3 miR-509-3p alteration of 3.87E−04 0.00869127 COL1A1; COL3A1; SPARC; YAP1/ECM axis WP3967 COL5A1; FN1; GPC6; TEAD1 Homologous recombination 4.94E−04 0.01059613 POLD3; POLD4; POLD1; WP186 POLD2; RPA1; ATM Nanoparticle-mediated activation 5.05E−04 0.01035965 ITGB1; COL1A1; NRAS; AKT3; of receptor signaling WP2643 ITGA1; FN1; MAPK1; KRAS; MAPK14 Benzo(a)pyrene metabolism 5.21E−04 0.01024099 AKR1C1; AKR1C3; AKR1A1; WP696 AKR1C2; CYP1B1 VEGFA-VEGFR2 Signaling 5.24E−04 0.00988413 YWHAE; ITGB1; ATF2; HDAC5; Pathway WP3888 ROCK1; SHC1; CLTC; CTNND1; IQGAP1; GJA1; ADAMTS1; PBK; CTNNA1; CCL2; MAPK1; RICTOR; IGFBP7; ITGAV; EPS15; EIF2A; NCK1; MAPK3; PRKCI; CAV1; MMP2; TRPC1; RPS6; RHOC; MAPK14; SOD2; DKK1; MMP14; PIK3CA; TXNIP; CTNNB1; ATF6; VCL Human Thyroid Stimulating 5.76E−04 0.01046413 RPS6; GNAI3; PLD1; MAPK14; Hormone (TSH) signaling GNAI1; GNAI2; RBL2; CCND3; pathway WP2032 GNG2; PIK3CA; CDK2; MAPK1; PLCB1; JAK1; MAPK3 TGF-beta Signaling Pathway 5.89E−04 0.01029146 ITGB1; ATF2; APP; KLF11; WP366 ROCK1; SHC1; CAV1; FN1; NEDD9; MAPK14; THBS1; PML; RBX1; PIAS1; TGFBR3; RBL2; KLF6; COPS5; COL1A2; SP1; CDK1; MAPK1; RNF111; SPTBN1 PDGFR-beta pathway WP3972 6.75E−04 0.01137075 PDGFRB; STAT5B; MAP3K1; PIK3CA; SHC1; RASA1; EIF2AK2; MAPK3; JAK1 Angiogenesis WP1539 7.88E−04 0.01282701 PDGFRA; ANGPT1; PIK3CA; TIMP2; ARNT; MAPK1; MAPK14; HIF1A Signal Transduction of S1P 0.00106913 0.01682102 AKT3; GNAI3; MAPK1; PLCB1; Receptor WP26 GNAI1; PIK3C2B; GNAI2; MAPK3 Complement and Coagulation 0.00150478 0.02291152 C1S; C1R; PROS1; CLTC; F2R; Cascades WP558 TFPI; SERPINA5; C3; LMAN1; C6; PROC; SERPING1; CD46 Prostaglandin Synthesis and 0.0016228 0.02393628 PTGFR; HPGD; ANXA2; Regulation WP98 PTGER2; AKR1C1; ANXA6; AKR1C3; S100A6; AKR1C2; PTGFRN; PTGS1 AGE/RAGE pathway WP2324 0.00176955 0.02530991 ATF2; STAT5B; MMP7; ROCK1; IRS1; SHC1; MMP2; MAPK14; HIF1A; LGALS3; MMP14; SP1; MAPK1; MAPK3 Disorders of the Krebs cycle 0.00177113 0.02458746 SUCLA2; DLST; SUCLG2; WP4236 DHTKD1 Epithelial to mesenchymal 0.00181147 0.02442895 NOTCH3; SPARC; SHC1; transition in colorectal cancer HIF1A; LRP6; AKT3; MAPK1; WP4239 MAPK3; FZD1; TGFB2; MMP2; WNT5A; FZD6; FN1; MAPK14; MPP5; LATS2; COL4A2; PIK3CA; COL4A1; CTDSP1; COL4A4; COL4A6; COL4A5; CTNNB1; KRAS Hippo-Merlin Signaling 0.00206112 0.0270236 PDGFRB; ITGB1; PDGFRA; Dysregulation WP4541 PPP1R12A; ITGA4; ITGA1; RBX1; PPP1CB; PPP1CC; NRAS; LATS2; PRKAR1A; ITGA10; CTNNA1; CDH24; CDH13; CTNNB1; ITGAV; KRAS; TEAD1; PAK4 Signaling Pathways in 0.00217158 0.02770228 PDGFRB; PDGFRA; PRKCI; Glioblastoma WP2261 IRS1; ARAF; PTEN; PIK3C2B; NRAS; PIK3CA; AKT3; ERBB2; CDK2; MAPK1; ATM; KRAS; MAPK3 Alzheimers Disease WP2059 0.00247196 0.03070432 APP; MME; LRP1; ADAM10; TNFRSF1A; BACE1; APH1A; CASP7; NCSTN; MAPK1; CALM3; CAPN1; APOE; PLCB1; ATF6; MAPK3 Leptin signaling pathway WP2034 0.00262103 0.03172115 NCOA1; STAT5B; ROCK1; IRS1; SHC1; RPS6; PTEN; MAPK14; SP1; CFL2; ERBB2; LEPR; MAPK1; JAK1; MAPK3 Proteasome Degradation WP183 0.00283608 0.03346579 UBA7; RPN2; RPN1; HLA-B; HLA-C; HLA-A; HLA-F; HLA-E; PSMB6; PSMB4; PSMB5; UBB; PSMD5 The human immune response to 0.00302587 0.03483445 MED14; IFNGR1; STAT2; tuberculosis WP4197 IFNGR2; IFIT1; PIAS1; JAK1 MAPK Cascade WP422 0.00307588 0.03456708 NRAS; MAP3K1; RRAS; ARAF; MAPK1; KRAS; MAPK14; MAPK3 Spinal Cord Injury WP2431 0.00380629 0.04178063 IL1R1; RHOC; AQP1; RHOB; FKBP1A; LGALS3; GJA1; VCAN; COL4A1; PTPRA; CDK2; CCNG1; CDK1; CCL2; MAPK1; CD47; VIM; ROS1; TLR4; MAPK3 Extracellular vesicle-mediated 0.00387145 0.0415301 TGFBR3; NRAS; TGFB2; signaling in recipient cells ERBB2; WNT5A; CTNNB1; WP2870 KRAS; MFGE8 Pathways up-regulated in HMLE-TWIST cells two days after chaetocin removal (@150 μM) Photodynamic therapy-induced 3.87E−06 0.00182518 IL1A; IL6; CSF2; CCND1; NF-kB survival signaling WP3617 MMP1; IL1B; MMP3; PTGS2; VEGFA; BIRC3 Photodynamic therapy-induced 2.73E−05 0.00643359 BCL2L11; WARS; EDEM1; unfolded protein response DDIT3; DNAJB11; ASNS; WP3613 TRIB3; ATF3 mRNA Processing WP411 1.13E−04 0.01782893 NCBP1; FUS; CSTF3; CPSF3; CSTF2; DDX20; U2AF1; DICER1; HNRNPAB; PPM1G; HNRNPM; NXF1; SFPQ; PRPF6; POLR2A; SNRPA1; SNRPF Pathways up-regulated in HMLE-TWIST cells two days after chaetocin removal (@500 μM) No pathway was identified that met the 0.05 p value threshold.
[0425] After the RNA-seq samples were sequenced, the cells used in this experiment were found to have a mycoplasma infection. Table 8 lists all the biological pathways up- or down-regulated in response to two days of chaetocin treatment. The results for two representative chaetocin concentrations (150 μM and 500 μM) are shown. Other concentrations produced a highly similar list of pathways. Only pathways whose enrichment is associated with an adjusted p value of less than 0.05 are indicated. Pathways listed in italicized font are those which have a known role in promoting the formation of the mesenchymal state in published literature. The pathways marked with an asterisk in tables above are those having known roles as tumor suppressors.
[0426] Table 9 lists biological pathways up- or down-regulated two days after chaetocin removal. The results for two representative chaetocin concentrations (150 μM and 500 μM) are shown. Other concentrations produced a highly similar list of pathways. Only pathways whose enrichment is associated with an adjusted p value of less than 0.05 are indicated. Pathways listed in italicized font are those which have a known role in promoting the formation of the mesenchymal state in published literature. Pathways marked with an asterisk in the above tables are those having known roles as tumor suppressors.
[0427] These data reveal that (1) chaetocin treatment inactivates several pathways which are critical for maintaining the mesenchymal, TIC state, (2) several tumor suppressor pathways are activated in response to chaetocin treatment, and (3) the chaetocin effect persist even after its removal. Together these data demonstrate that chaetocin treatment results in an epigenetic change which reprograms mesenchymal, TICs into non-TICs.
Example 12. Co-Treatment with Chaetocin and Temozolomide Sensitizes a Patient-Derived Temozolomide-Resistant Glioblastoma Cell Line to Temozolomide Treatment
[0428] A short exposure to chaetocin is sufficient to cause an epigenetic change, reprogramming mesenchymal, TIC-rich cancer cells into an epithelial fate while sensitizing them to chemotherapy. Considering epigenetic changes are stable and can continue in the absence of the initial treatment, chaetocin treatment effects (mesenchymal reprogramming and sensitization to chemotherapy) should occur even if chaetocin is administered at the same time as the chemotherapy. To test this, a patient-derived Temozolomide (TMZ)-resistant glioblastoma (GBM) cell line was grown in suspension into spheroids and treated with chaetocin, TMZ, or both (administered at the same time). To clarify, when TMZ and chaetocin were administered together, they were added at the same time to the spheroids. Cell confluence, an assay for spheroid size, was measured to determine if cell growth was inhibited (flat curve) or spheroid sizes were reduced (indicator of cell death and spheroid shrinkage).
[0429] This effect was tested against a range of TMZ concentrations.
[0430] Based on these data we claim that (1) chaetocin treatment works effectively to sensitize chemotherapy resistant GBM cells to chemotherapy; (2) administration of chaetocin together with TMZ is effective at sensitizing GBM cells to chemotherapy.
OTHER EMBODIMENTS
[0431] Various modifications and variations of the described disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the art are intended to be within the scope of the disclosure.
[0432] Other embodiments are in the claims.