SLFN11 AS BIOMARKER FOR AML

Abstract

The use of Slfn11 as a biomarker for detecting the occurrence of epithlial-to-mesenchymal transition (EMT) in a subject, and the use of Slfn11 modulators to treat cancer is disclosed herein. Also disclosed are various methods for detecting the occurrence of epithelial-to-mesenchymal transition (EMT) in a subject by measuring Slfn11 expression and/or activity.

Claims

1. A method of identifying a subject having an Axl-related condition, the method comprising assessing the level of expression or activity of Slfn11 in the subject, or in a sample derived from the subject.

2. A method according to claim 1 of identifying a subject having a particular risk of developing metastatic or drug-resistant cancer, the method comprising assessing the level of expression or activity of Slfn11 in the subject, or in a sample derived from the subject, an increased level of Slfn11 expression or activity indicating an increased risk of the subject of developing metastatic or drug-resistant cancer.

3. A method according to claim 1 of identifying the presence of a Cancer Stem Cell in a subject, the method comprising determining the level of Slfn11 expression or activity in the subject, or in a sample derived from the subject, increased expression or activity of Slfn11 indicating the existence of a Cancer Stem Cell (CSC).

4. A method according to claim 1 of identifying a subject undergoing epithelial-to-mesenchymal transition (EMT), the method comprising determining the level of Slfn11 expression or activity in the subject, or in a sample derived from the subject, an increase in expression or activity of Slfn11 indicating the occurrence of EMT.

5. A method of prognosing a cancer-related outcome in a subject, the method comprising assessing Slfn11 activity or expression in the subject, or in a sample derived from the subject.

6. A method according to claim 5, wherein: (i) an increase in Slfn11 activity or expression relative to a control sample is indicative of susceptibility to treatment with an agent capable of inhibiting or reversing EMT, or of increased susceptibility to a chemotherapeutic agent; (ii) a decrease in Slfn11 activity or expression relative to a control sample is indicative of resistance to treatment with an agent capable of inhibiting or reversing EMT, or of reduced susceptibility to a chemotherapeutic agent; (iii) a decrease in Slfn11 activity or expression relative to a control sample following treatment of the subject with an Axl or Akt3 inhibitor is indicative of susceptibility to treatment with an agent capable of inhibiting or reversing EMT, or of increased susceptibility to a chemotherapeutic agent; or (iv) an increase in Slfn11 activity or expression relative to a control sample following treatment of the subject with an Axl or Akt3 inhibitor is indicative of resistance to treatment with an agent capable of inhibiting or reversing EMT, or of reduced susceptibility to a chemotherapeutic agent.

7. A method according to claim 6, wherein the agent capable of inhibiting or reversing EMT is an Axl inhibitor, Akt3 inhibitor, or Slfn11 inhibitor.

8. A method of identifying Axl activity, the method comprising determining the level of Slfn11 expression or activity in the subject, or in a sample derived from the subject, increased expression or activity of Slfn11 correlating with Axl activity.

9. A method according to any one of claims 1 to 8 in which the subject is mammalian.

10. A method according to claim 9 in which the subject is human.

11. A method according to any one of claims 1 to 10, wherein the level of expression or activity in the subject or sample derived from the subject is determined relative to a control sample.

12. A method according to any one of claims 1 to 11, wherein the level of expression of Slfn11 is assessed by determining the copy number of the gene encoding Slfn11 relative to a control sample, wherein an increase in the copy number indicates an increased level of expression of Akt3.

13. A method according to any one of claims 1 to 12, wherein the level of expression of Slfn11 is assessed by determining the level of Slfn11 protein or mRNA.

14. A method of selecting patients, preferably human patients, for treatment of an Axl-related condition, the method comprising identifying patients having elevated Slfn11 activity or expression and selecting thus identified patients for treatment.

15. A method of selecting patients according to claim 14 in which the Axl-related condition is cancer.

16. A method according to any one of claim 14 or 15, wherein the patient is identified according to a method of any one of claims 1 to 13.

17. A method according to any one of claims 14 to 16, wherein the treatment comprises administering an agent capable of inhibiting or reversing EMT.

18. A method according to claim 17, wherein the agent comprises a Slfn11 inhibitor, an Akt3 inhibitor, or an Axl inhibitor.

19. A method according to any one of claims 1 to 23, wherein the cancer or Axl-related condition is a cancer selected from acute myelocytic leukemia (AML), breast, melanoma, prostate, ovarian, colorectal, lung or glioma cancer.

20. An Slfn11 modulator for use in the treatment of an Axl-related condition.

21. An Slfn11 modulator according to claim 20 in which the condition is cancer.

22. An Slfn11 modulator for use in the inhibition of EMT.

23. A compound capable of modulating Slfn11 activity for use in the prevention, inhibition, or treatment of drug resistance in a subject having cancer, the method comprising contacting the subject with a compound capable of modulating Slfn11 activity or expression.

24. A Slfn11 modulator according to any one of claims 21 to 23 in combination with another therapeutic agent.

25. A Slfn11 modulator according to any one of claims 20 to 24, wherein the modulator in an Slfn11 inhibitor.

26. A method of treating a subject having an Axl-related condition, the method comprising contacting the subject with an Slfn11 modulator or pharmaceutical compound selected as, or derived from, a candidate compound obtained by a method according to any one of claims 38 to 42.

27. A method of treatment of a subject according to claim 26 having an Axl-related condition, the method comprising periodically assessing Slfn11 activity or expression in the subject.

28. A method according to one of claims 26 to 27 in which the Axl-related condition is cancer.

29. A method according to one of claims 26 to 28 in which treatment of the subject is adjusted according to detected levels of Slfn11 activity or expression.

30. A method according to any one of claims 26 to 29 in which the subject is being treated with a Slfn11 inhibitor, a Slfn11 activator, an Axl inhibitor, or an Akt3 inhibitor.

31. A method of inhibiting EMT in a subject, the method comprising contacting the subject with a compound capable of inhibiting Slfn11 activity or expression.

32. A method of inhibiting Cancer Stem cells in a subject, the method comprising contacting the subject with a compound capable of inhibiting Slfn11 activity or expression.

33. A method according to any one of claims 26 to 32 in which the subject is also contacted with another cancer therapeutic.

34. A method of preventing or inhibiting drug resistance in a subject having cancer, the method comprising contacting the subject with a compound capable of modulating Slfn11 activity or expression.

35. A method according to any one of claims 26 to 34 in which the subject is mammalian.

36. A method according to claim 35 in which the subject is human.

37. An Slfn11 modulator according to any one of claims 20 to 25, or a method of treatment according to any one of claims 33 to 36 in which the other therapeutic agent is a cancer treatment selected from alkylating agents, including alkyl sulfonates such as busulfan, nitrogen mustards such as chlorambucil, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, and uramustine, ethyleneimine derivatives such as thiotepa, nitrosoureas such as carmustine, lomustine, and streptozocin, triazenes such as dacarbazine, procarbazine, and temozolamide, platinum compounds such as cisplatin, carboplatin, oxaliplatin, satraplatin, and picoplatin onnaplatin, tetraplatin, sprioplatin, iproplatin, chloro(diethylenediamino)-platinum (II) chloride, dichloro(ethylenediamino)-platinum (II), diamino(2-ethylmalonato)platinum (II), (1,2-diaminocyclohexane)malonatoplatinum (II), (4-carboxyphthalo)-(1,2-diaminocyclohexane)platinum (II), (1,2-diaminocyclohexane)-(isocitrato)platinum (II), and (1,2-diaminocyclohexane)-cis-(pyruvato)platinum (II); antimetabolites, including antifolates such as methotrexate, permetrexed, raltitrexed, and trimetrexate,pyrimidine analogues such as azacitidine, capecitabine, cytarabine, edatrexate, floxuridine, fluorouracil, gemcitabine, and troxacitabine, and purine analogues such as cladribine, chlorodeoxyadenosine, clofarabine, fludarabine, mercaptopurine, pentostatin, and thioguanine; natural products, including antitumor antibiotics such as bleomycin, dactinomycin, mithramycin, mitomycin, mitoxantrone, porfiromycin, and anthracyclines such as daunorubicin, doxorubicin, epirubicin, idarubicin, and valrubicin, mitotic inhibitors such as the vinca alkaloids vinblastine, vinvesir, vincristine, vindesine, and vinorelbine, enzymes such as L-asparaginase and PEG-L-asparaginase, microtubule polymer stabilizers such as the taxanes paclitaxel and docetaxel, topisomerase I inhibitors such as the camptothecins irinotecan and topotecan, and topoisomerase II inhibitors such as podophyllotoxin, amsacrine, etoposide, teniposide, losoxantrone and actinomycin; hormones and hormone antagonists, including androgens such as fluoxymesterone and testolactone, antiandrogens such as bicalutamide, cyproterone, flutamide, and nilutamide, corticosteroids such as dexamethasone and prednisone, aromatase inhibitors such as aminoglutethimide, anastrozole, exemestane, formestane, and letrozole: estrogens such as diethylstilbestrol, antiestrogens such as fulvestrant, raloxifene, tamoxifen, and toremifine, luteinising hormone-releasing hormone (LHRH) agonists and antagonists such as abarelix, buserelin, goserelin, leuprolide, histrelin, desorelin, nafarelin acetate and triptorelin, progestins such as medroxyprogesterone acetate and megestrol acetate, and thyroid hormones such as levothyroxine and liothyronine; PKB pathway inhibitors, including perifosine, enzastaurin hydrochloride, and triciribine, P13K inhibitors such as semaphore and SF1126, and MTOR inhibitors such as rapamycin and analogues; CDK inhibitors, including seliciclib, alvocidib, and 7-hydroxystaurosporine; COX-2 inhibitors, including celecoxib; HDAC inhibitors, including trichostatin A, suberoylanilide hydroxamic acid, and chlamydocin; DNA methylase inhibitors, including temozolomide, and miscellaneous agents, including altretamine, arsenic trioxide, thalidomide, lenalidomide, gallium nitrate, levamisole, mitotane, hydroxyurea, octreotide, procarbazine, suramin, photodynamic compounds such as methoxsalen and sodium porfimer, and proteasome inhibitors such as bortezomib: molecular targeted therapy agents including: functional therapeutic agents, including gene therapy agents, antisense therapy agents,tyrosine kinase inhibitors such as erlotinib hydrochloride, gefitinib, imatinib mesylate, and semaxanib, Raf inhibitors such as sorafenib, and gene expression modulators such as the retinoids and rexinoids, for example adapalene, bexarotene, trans-retinoic acid, 9-cis-retinoic acid, and N-(4-hydroxyphenyl)retinamide; and phenotype-directed therapy agents, including monoclonal antibodies such as alemtuzumab, bevacizumab, cetuximab, ibritumomab tiuxetan, rituximab, and trastuzumab, immunotoxins such as gemtuzumab ozogamicin, radioimmunoconjugates such as I-tositumobab, and cancer vaccines; Biologic therapy agents including: interferons such as interferon-[alpha]2a and interferon-[alpha]2b, and interleukins such as aldesleukin, denileukin diftitox, and oprelvekin anticancer therapies involving the use of protective or adjunctive agents, including:cytoprotective agents such as amifostine, and dexrazoxane, phosphonates such as pamidronate and zoledronic acid, and stimulating factors such as epoetin, darbeopetin, filgrastim, PEG-filgrastim, and sargramostim; and Axl inhibitor such as 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7-(S)-pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; or further combination chemotherapeutic regimens, such as combinations of carboplatin/paclitaxel, capecitabine/docetaxel, fluorauracil/levamisole, fluorauracil/leucovorin, methotrexate/leucovorin, and trastuzumab/paclitaxel, alone or in further combination with carboplatin, and the like.

38. A method of selecting a pharmaceutical compound useful for the prevention, inhibition or treatment of an Axl-related condition, the method comprising providing a group of candidate pharmaceutical compounds for testing, testing the effect of candidate pharmaceutical compounds on Slfn11 activity or expression in a test system, and selecting a candidate pharmaceutical compound on the basis of modulating Slfn11 activity or expression.

39. A method of selecting a candidate pharmaceutical compound useful in the treatment of metastatic or drug resistant cancer, the method comprising providing a group of candidate pharmaceutical compounds for testing, testing the effect of candidate pharmaceutical compounds on Slfn11 activity or expression in a test system, and selecting a candidate pharmaceutical compound on the basis of its modulation of Slfn11 activity or expression.

40. A method of selecting a candidate pharmaceutical compound useful in the prevention or inhibition of EMT, the method comprising providing a group of candidate pharmaceutical compounds for testing, testing the effect of candidate pharmaceutical compounds on Slfn11 activity or expression in a test system, and selecting a candidate pharmaceutical compound on the basis of modulating Slfn11 activity or expression.

41. A method of selecting a candidate pharmaceutical compound useful in the prevention, inhibition or treatment of an Axl-related condition, the method comprising selectively reducing expression of Slfn11 in a test cell, contacting the test cell with the candidate pharmaceutical compound and determining the effect of the candidate pharmaceutical compound on the modulation of Slfn11 activity or expression.

42. A method of selecting a candidate pharmaceutical compound useful in the prevention, inhibition or treatment of an Axl-related condition, the method comprising selectively reducing expression of Slfn11 in an in vitro test system to a low level contacting the test system with a candidate pharmaceutical compound, and selecting candidate pharmaceutical compounds which modulate Slfn11 activity or expression.

43. A method according to any one of claims 38 to 42 in which candidate pharmaceutical compounds which substantially or completely inhibit Slfn11 activity or expression are selected.

44. A method of selecting candidate pharmaceutical compounds according to claim 41, 42 or 43 in which inhibition of Slfn11 activity or expression is indicated by a reduction in EMT.

45. A method according to any one of claims 40 to 44 in which the expression of Slfn11 in cells in the test system is reduced by 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20%.

46. A method according to claim 45 in which the expression of Slfn11 is reduced so as to not cause inhibition of EMT.

47. A method according to any one of claims 40 to 46 in which the expression of Slfn11 is selectively reduced by introducing into cells in the test system with a nucleotide which interferes with expression of Slfn11.

48. A cell line which is sensitive to inhibitors of EMT, the cell line having a level of Slfn11 expression that is just insufficient to prevent EMT.

49. A cell line according to claim 48 which is a human cell line.

50. A method of identifying a compound which inhibits Slfn11 activity or expression, the method comprising contacting a cell from a cell line according to claim 48 or 49 with a test compound and determining inhibition of Slfn11 activity or expression in the cell.

51. A method according to claim 50 in which inhibition of Slfn11 activity or expression is identified by inhibition of EMT.

52. Use of Slfn11 as a biomarker for detecting the occurrence of epithelial-to-mesenchymal transition (EMT) in a subject.

53. Use according to claim 52 wherein an increase in the expression and/or activation of Slfn11 is indicative of the occurrence of epithelial-to-mesenchymal transition (EMT).

54. Use of Slfn11 as a biomarker for detecting the expression and/or activation of Axl, wherein an increase in the expression and/or activation of Slfn11 is indicative of an increase in the expression and/or activation of Axl.

55. A method for detecting the occurrence of epithelial-to-mesenchymal transition (EMT) in a sample, said method comprising determining the expression level or activation of Slfn11 in a sample isolated from a cell, group of cells, an animal model or human as compared to a control sample, wherein an increase in the expression level or activation of Slfn11 relative to the control sample is indicative of the occurrence of epithelial-to-mesenchymal transition (EMT).

56. A method for identifying an agent capable of inhibiting or reversing epithelial-to-mesenchymal transition (EMT), said method comprising administering said agent to a cell, group of cells or animal model, and monitoring the activation and/or the expression of Slfn11.

57. A method according to claim 56 which comprises: (i) administering the agent to a cell, group of cells or an animal model, not a human; and (ii) measuring Slfn11 expression and/or Slfn11 activation in samples derived from the treated and the untreated cells or animal model; and (iii) detecting an increase in the expression and/or activation of Slfn11 in the treated sample as compared to the untreated sample as an indication of the ability to inhibit or reverse epithelial-to-mesenchymal transition (EMT).

58. A method according to claim 56 or claim 57, wherein the animal model is not a human.

59. A use or method according to any one of claims 53 to 58 wherein the level of expression of Slfn11 is assessed by determining the copy number of the gene encoding Slfn11 relative to a control sample, wherein an increase in the copy number indicates an increased level of expression of Slfn11.

60. A use or method according to any one of claims 52 to 59 wherein the level of expression of Slfn11 is assessed by determining the level of Slfn11 protein or mRNA.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0176] FIG. 1 shows a single gene graph describing regulation of SLFN11 in response to 14d BGB324-treatment in mice bearing subcutaneous MV4-11 xenografts. The transcript is 3.98-fold decreased from control-level in the tumors from mice treated with the highest dose (50 mg/kg);

[0177] FIG. 2 shows a comparison of the expression levels of Axl and SLFN11. The comparison demonstrates that these two proteins co-localize in normal blood myeloid cells (red circle);

[0178] FIG. 3 shows Slfn11 expression in PBMCs. The mouse anti-human Slfn11 antibody was diluted 1:50-1:600 and used for staining of fixed PBMCs. Figure shows representative histograms of stained PBMCs compared to secondary antibody/unstained control. Geometric mean is calculated based on fluorescent measurements of 50 000 cells. Dotplots show Slfn11 positive cells in 3 different blood subpopulations;

[0179] FIG. 4 shows Slfn11 and Axl coexpression in healthy blood. A. Flow cytometry scatter plot of Axl (FL-4 ch) and Slfn11 (F1-1 ch) stained PBMCs from healthy volunteers. Left panel (A01)) is unstained sample and the Q1-4 gate has been set according to this sample. Middle panel (A05) is secondary ab control which function as a control to verify that all negative cells are located in quadrant 4 (Q4). Right panel (A03) is Axl/Slfn11 costained sample showing Axl+ cells (Q1), Axl+/Slfn11+ cells (Q2), Slfn11+ cells (Q3) and negative cells (Q4). B. Backgating of samples showing where the stained samples are localized in the viable PBMC population. Left panel is Axl+ cells, middle panel is Axl+/Slfn11+ cells whereas right panel is Slfn11+ cells only;

[0180] FIG. 5 shows Slfn11 expression in AML cell lines. Western blot analysis (A) showing Slfn11 expression levels in inhouse AML cell lines MOLM-13, OCI-M1, OCI-AML3 and Kasumi cells. HeLa cell lysate serve as a negative control.

The Slfn11 expression levels in these cells were confirmed by flow cytometry (B) using the same Slfn11 primary antibody and AlexaFluor488 conjugated secondary antibody. Data is representative for 2 experiments (10 000 cells per measurement);

[0181] FIG. 6 shows a western blot of MV4-11 xenografts from mice treated for 14 days with 25 or 50 mg/kg, or with a single high dose (100 mg/kg) of BGB324.

[0182] FIG. 7 shows a western blot of MV4-11 xenografts from mice treated with a single low (50 mg/kg, upper panels) or high (100 mg/kg, lower panels) dose of BGB324. The tumors were harvested at different time points after treatment, from 4-up to 72 hours.

[0183] FIG. 8 shows the reduction of SLFN11 expression levels after BGB324 treatment. AML cell lines were treated with BGB324 (IC50 values) for 24 h, 48 h and 72 h and analysed by flow cytometry. A representative flow histograms for MOLM-13, OCI-AML3 and OCI-M1 after treatment for 24 h (upper panels), 48 h (mid panels) and 72 h (lower panels) showing control cells (red), BGB324 treated cells (blue), secondary antibody control (green) and unstained sample (pink). B. Flow cytometric analysis of Slfn11 expression after BGB324 treatment of Kasumi (n=1), OCI-M1 (n=2), OCI-AML3 (n=2), OCI-AML5 (n=1) and MOLM-13 wt (n=3), MOLM-13shLuc (n=1) and MOLM-13shAxl (n=2). Data show geometric mean (% of control+−S.D. of 2-3 experiments.

[0184] FIG. 9 shows the results of treatment with BGB324 show a dose-dependent downregulation of Slfn11 in MOLM-13 wt cells. MOLM-13 cells were treated with BGB324 for 48 hours at concentrations ranging from 0.1 uM to 1.2 uM. Slfn11 expression levels were analysed by western blot (A) and flow cytometry (B) using mouse anti-human Slfn11 antibody.

[0185] FIG. 10 shows the response of total SLFN11 levels in AML cell lines after 24, 48 and 72 hours of treatment with BGB324 at 0.05, 0.1 or 0.3 μM (MOLM13 and Mv4-11, upper panels) or at 2.5 μM (Kasumi and OCI-AML5, lower panels). The graphs show geometric mean of fluorescence, calculated as % of control (which is set to 100%—indicated by a dotted line), ±SEM. * indicates significance relative to control, calculated using a two-tailed Student's t-test. * p<0.05, **p<0.005, n=3.

[0186] FIG. 11 shows spleen, bone marrow and blood from mice stained with anti-human-CD33 and -CD45 antibodies to identify leukemic cells in the tissues. CD33/CD45 double-positive cells were quantified as % of total live cell count (A). Bone marrows and spleens from treated and non-treated mice were assessed for biomarker expression by flow cytometry. The samples were stained with anti-human CD33 antibody, and biomarker expression was only evaluated in CD33-positive cells (B). The graphs show geometric mean of fluorescence, calculated as % of control (which is set to 100%), ±SEM. * indicates significance relative to control, calculated using a two-tailed Student's t-test. * p<0.05, **p<0.005, n>5.

MATERIAL AND METHODS

Materials

[0187] 10% NuPAGE® Bis-Tris precast gels (#NP0301BOX, Invitrogen) [0188] Amersham Hybond-P PVDF transfer membrane (#RPN303F, GE Healthcare) [0189] BGB324 (Manufacturer: Almac Group, N Ireland. Lot #011-SR-324 DA2aI-15, 10 mM in DMSO, prepared 17.02.14 by Tone Sandal) [0190] BD Phosflow™ Lyse/Fix Buffer 5× (#558049, BD Biosciences) [0191] Complete Mini Protease Inhibitor Cocktail tablets (#04693116001, Roche) [0192] ECL-reagents: Reagent1 and Reagent2 (#1859701 and #1859698, Thermo Scientific) [0193] Fetal Bovine Serum (FBS, #A9647, Sigma) [0194] MagicMark™ XP Western Protein Standard (#LC5602, Invitrogen) [0195] Mouse-anti-human Axl Ab (1H12-1B7-5D6, BerGenBio, BGB #47) [0196] Mouse-anti-human Axl Ab (1H12-1B7-5D6, BerGenBio, BGB #47) Alexa 647-conjugated (1.2 mg/ml in PBS. Stock: 22 Oct. 2013, made by Hallvard Haugen) [0197] Mouse-anti-human SLFN11 AB (sc-374339, Santa Cruz, BGB #91) [0198] Nitrocellulose membrane, Whatman Protran BA85 (#10401196, GE Healthcare) [0199] NP-40: Pierce IP lysis buffer (#87788, Thermo Scientific) [0200] NuPAGE Antioxidant (#NP0005, Invitrogen) [0201] NuPAGE LDS Sample Buffer 4× (#NP0007, Invitrogen) [0202] phosSTOP Phosphatase Inhibitor Cocktail tablets (#04693116001, Roche) [0203] Pierce BCA protein assay kit (#PI-23227, Thermo Scientific) [0204] Rabbit anti actin Ab (#A5060, Sigma-Aldrich) [0205] Rabbit anti-human GAPDH [EPR6256] AB (#ab128915, Abcam) [0206] SeeBlue® Plus 2 Pre-Stained Standard (#LC5625, Invitrogen) [0207] AlexaFluor 488 goat anti-mouse IgG (H+L) (#A11029, Invitrogen) [0208] Goat anti-mouse IgG (H+L), Horseradish peroxidase conjugate (#G-21040, Life Technologies)

Cells

[0209] MOLM13 cells were grown in RPMI-1640 media (R8758, Sigma-Aldrich), supplemented with 10% fetal bovine serum (FBS), L-glutamine (4 mM) and penicillin-streptomycin (5 μg/ml).

[0210] Mv4-1/cells (ATCC, CRL9591) were grown in Isovec s Modified Dulbecco's Medium (IMDM; #30-2005, ATCC) supplemented with 10% fetal bovine serum (FBS), L-glutamine (4 mM), streptomycin (5 μg/ml) and penicillin (5 U/ml).

[0211] OCI-M1 cells were grown in were grown in Isovec's Modified Dulbecco's Medium (IMDM: #30-2005, ATCC) supplemented with 5% fetal bovine serum (FBS), L-glutamine (4 mM), streptomycin (5 μg/ml) and penicillin (5 U/ml).

[0212] OCI-AML3 cells were grown in Alpha MEM (#22561-021, Gibco by Life Technologies) supplemented with 20% fetal bovine serum (FBS L-glutamine (4 mM), streptomycin (5 μg/ml) and penicillin (5 U/ml).

[0213] OCI-AML5 cells were grown in Alpha MEM (#22561-021, Gibco by Life Technologies) supplemented with 20% fetal bovine serum (FBS), L-glutamine (4 mM), streptomycin (5 μg/ml), penicillin (5 U/ml), and GM-CSF (2.5 ng/ml).

[0214] Kasumi cells were grown in RPMI-1640 media (R8758, Sigma-Aldrich) supplemented with 20% fetal bovine serum (FBS), L-glutamine (4 mM), streptomycin (5 μg/ml) and penicillin (5 U/ml).

Methods

Western Blot, General Protocol

[0215] For western blot analysis, cells were lysed on ice using NP-40 lysis buffer with protease- and phosphatase inhibitors. Total protein concentration in lysates was measured using a BCA protein assay kit following the manufacturers instructions. 10% NuPAGE® Bis-Tris precast gels were loaded with 30-50 μg of protein in each well diluted in sample buffer and antioxidant. A 1:1 mix of MagicMark™ XP Western Protein Standard (Invitrogen) and SeeBlue® Plus 2 Pre-Stained Standard was used as protein standard. Gels were run at 50V for 20 min, then at 100V for 1 h30 min. Blotting was done on ice for 1 h 30 min at 100V onto PVDF (pre-activated with MeOH) or nitrocellulose membranes. Membranes were washed in TBS-0.1% Tween-20 (TBS-T), and blocked in TBS-T 5% BSA for at least 1 h at RT. Primary antibody was added at 1:1000 in TBS-T 5% BSA (rabbit-anti-actin Ab was added at 1:2000, rabbit anti-GAPDH Ab was added at 1:3000) in TBS-T 5% BSA, and membranes were incubated over night at 4° C. Membranes were then washed 3× in TBS-T, and incubated in HRP-conjugated secondary antibody at 1:5000 dilution in TBS-T 5% milk for 45 min at RT. Membranes were developed for 1 min using ECL-reagents and imaged with chemiluminescence using a Molecular Imager ChemiDoc™ XRS (BioRad).

[0216] All incubation- and washing steps were done on a roller.

Staining of Cells for Flow Cytometry, General Protocol

[0217] Live cells were centrifuged at 300 g for 5 minutes, washed once with PBS and centrifuged again. Cells were then fixed in 4% PFA in PBS for 10 minutes at 37° C., and resuspended in PBS. Unless processed immediately, samples were at this point stored in PBS at 4° C. (up to three weeks) or at −80° C. (for long-term storage). If intracellular epitopes were stained, cells were permebealized in 90% MeOH for 30 minutes on ice. Unless processed immediately, samples were also be stored in 90% MeOH at −20° C. for up to two months.

Further Staining Procedure (Permebealized or Non-Permebealized Cells)

[0218] Cells (150-200 000 per sample) were washed 1× in PBS and incubated in blocking buffer; (PBS+0.5% BSA) for 15 minutes at room temperature. Thereafter, cells were incubated with primary mouse-anti-human SLFN11 Ab at the indicated dilutions in incubation buffer (IB, PBS+0.5% BSA) for 1 hour at room temperature. Cells were then washed 3× with IB and incubated with secondary AB (conjugated to a fluorescent flurophore) at 1:1000 dilution in IB for 30 minutes at room temperature. Finally, cells were washed 3× in IB and resuspended in PBS. For costaining experiments, cells were subsequently incubated with mouse-anti-human Axl Ab (1H12-A647 conjugated Ab) diluted 1:3000 in IB. Cells were analyzed immediately, or stored for up to 24 hours at 4° C. before analysis.

[0219] Analysis of cells was done on a BD LSR Fortessa or a BD C6 Accuri flow cytometer, and further processing was done using FlowJo v.7.6.

[0220] All incubation steps were done on a spinning wheel or gentle shaker.

Staining of Blood for Flow Cytometry, General Protocol

[0221] Human blood from healthy donors was collected in the presence of sodium citrate and mixed with 20 volumes of pre-warmed BD phosflow lyse/fix buffer (diluted to 1× in distilled water), followed by incubation in 37° C. water bath for 10 minutes. Cells were spun at 500 g for 8 minutes and washed once with PBS. Cells were permeabilized by adding 70% MeOH followed by 30 min incubation on ice. Unless processed immediately, samples were at this point stored in MeOH at −20° C. for up to 4 weeks.

[0222] Prior to antibody staining, cells were spun at 600 g, washed twice and resuspended in IB buffer. Cells were aliquoted into volumes corresponding to 100 ul collected blood (before dilution), and stained as described above (section two in “Staining of cells for flow cytometry, general protocol”).

[0223] All washing and incubation steps were done on a spinning wheel or gentle shaker.

EXAMPLES

Example 1: SLFN11 is Reduced in MV4-11 Xenografts after BGB324 Treatment

[0224] To identify new potential biomarkers of BGB324 efficacy in AML, microarray analyses of tumor material from several AML studies were performed. In the first two studies, NOD/SCID mice were implanted with subcutaneous Mv4-11 xenografts. In study 1 the mice were treated with control (vehicle) or a single dose of BGB324 (50 or 100 mg/kg), and sacrificed after 24 hours. In study 2 the mice were treated BID for 14 days with control (vehicle), low (25 mg/kg) or high (50 mg/kg) dose of BGB324 for 14 days. In a third study, MOLM13 cells were injected intravenously into the tail vein of NSG mice. After three days of inoculation, the animals were treated with control (vehicle), high (50 mg/kg) or low (25 mg/kg) dose of BGB324 QD until the vehicle-treated animals were moribund (14 days after initiation of treatment). Development of leukemia was confirmed by flow cytometric analyses of human CD33/CD45-positive cells in the blood, spleens and bone marrow, and RNA isolated from spleens were sent for microarray analysis.

[0225] One of the most interesting hits from a Rank Product Analysis of study 2 was a transcript called SLFN11. In this study, a highly significant dose-dependent downregulation of SLFN11 after 14d treatment was found (25 mg/kg: 1.98-fold downregulation, q=0, rank #3, 50 mg/kg: 3.98-fold downregulation, q=0, rank #9) (FIG. 3). A Rank Product Analysis of study 1 (single dose treatment) also revealed a significant reduction of SLFN11, although it was not one of the top hits in this dataset (1.449-fold, q=0, rank #245).

[0226] Furthermore, a SAM (significance of microarray) analysis of the same experiment (study 2) showed reduction of SLFN11 as the most significant (#1) hit (q=0.0) after BGB324 treatment. However, in the systemic AML study (study 3), SLFN11 was not among the transcripts that were significantly downregulated after BGB324-treatment.

Example 2: AXL and SLFN11 are Co-Expressed in Blood Myeloid Cells

[0227] The results from the microarray analyses strongly suggest that reduction of the SLFN11 transcripts in MV4-11 tumors is a direct consequence of Axl inhibition by BGB324. Thus, there might be crosstalk between these two proteins. However, there are currently no publications linking Axl and Slfn11 functionally. Therefore, it was decided to investigate the expression levels of these two proteins in different tissues using the GeneSapiens website (http://ist.medisapiens.com/, IST4 database). GeneSapiens offers a freely available fully integrated and annotated online database of the human transcriptome, where the expression levels of genes can be compared in healthy tissue as well as in tumor tissue and cell lines. A comparison of AXL and SFLN11 gene expression showed that AXL and SLFN11 has a high level of co-expression in normal blood myeloid cells (FIG. 2), suggesting that Axl and Slfn11 might also have a functional relationship in these cells.

Example 3: Slfn11 is Expressed in Various PBMC Subpopulations

[0228] To evaluate the result from the database comparison, the expression of Slfn11 and Axl was investigated in blood samples from healthy donors. Blood was collected from a healthy volunteer (id:G) in sodium citrate vacutainer. The blood was fixed and PBMCs were prepared for flow. As an initial experiment, Slfn11 mouse monoclonal antibody was tested on blood by performing a antibody dilution series in the range from 1:50-1:600. By flow cytometric analysis it was found that Slfn11 expression in various subpopulations of blood (FIG. 3), and dilution 1:100 was chosen as standard dilution for further experiments staining PBMCs.

[0229] Next, it was investigated if Slfn11 and Axl were coexpressed in any of the different blood subpopulations. PBMCs were prepared as above, stained with Slfn11 Ab (1:100) and secondary antibody Alexa488 (1:1000), followed by staining with 1H12-Alexa 647 conjugated antibody (1:3000). By flow cytometric analysis expression of both Axl and Slfn11 in PBMCs was found. However, coexpression of Axl and Slfn11 was only identified in a small subpopulation of cells (FIG. 4).

[0230] As seen in FIG. 4, it should be noted that coexpression of Slfn11 and Axl is mostly seen in the population which appears to be granulocytes, although the identity of these cells needs to be confirmed with immunocell-specific CD-marker staining (see discussion for further explanation).

Example 4: Slfn11 Expression in AML Cells Lines

[0231] After confirming the expression of Slfn11 in normal blood PBMCs, it was decided to further investigate the expression level of Slfn11 in AML cells. A panel of AML cell lines were examined both by flow cytometry and western blot. Western blot revealed Slfn11 expression in several of the AML lines, including MOLM13, OCI-M1 and OCI-AML3, but not in Kasumi cells (FIG. 5A). HeLa cells were included as a negative control, as these cells have been shown not to express SLFN11.

[0232] These results were subsequently confirmed by flow cytometry using the same antibody. As can be observed in FIG. 5B, flow cytometric analysis shows a low, but detectable level of Slfn11 in Kasumi cells, although this is not detected by Western Blotting (FIG. 5A).

Example 5: Slfn11 Expression is Reduced in MV4-11 Tumor Xenografts after BGB324 Treatment

[0233] The data from microarray analyses of MV4-11 tumor xenografts showed a significant reduction of the SLFN11 transcripts in the tumor cells after both long-term treatment as well as treatment with only a single dose of BGB324. To confirm whether this also resulted in a subsequent reduction of the Slfn11 protein in these tumors, lysates of MV4-11 subqutaneous tumor xenografts from both single dose and continuous treatment were examined by western blot. In these studies, mice had been treated with BGB324 25 mg/kg BID and 50 mg/kg BID 2-5 cycle for 14 days, or with a single dose 100 mg/kg (tumors harvested 6 h after treatment). In these tumors, a marked reduction of Slfn11 compared to control levels was found after 14 days of treatment. In tumors from mice treated with only a single high (100 mg/kg) dose of BGB324, Slfn11 was reduced in one of the two parallel samples examined (FIG. 6).

[0234] Since Slfn11 was reduced in only one of two parallel tumor samples after a single treatment, it was decided to examine more lysates from Study 1 by western blot. In this study, mice with subcutaneous MV4-11 tumor xenografts were treated with a single low (50 mg/kg) or high (100 mg/kg) dose of BGB324, and tumors were harvested at different time points, from 4 hours up to 72 hours after treatment. A slight reduction of Slfn11 could be detected 72 hours after treatment in mice treated with a low dose (50 mg/kg) of BGB324 (FIG. 7, upper panels). In mice treated with a high dose (100 mg/kg) of BGB324, reduction of Slfn11 in tumors 24, 48 and 72 hours after treatment was observed (FIG. 7, lower panels), but not at earlier time points (unlike the results from FIG. 6). Thus, it appears that Slfn11 is reduced in MV4-11 xenografts within 24-72 hours (depending on the drug concentration) after treatment with a single dose of BGB324. The effect of BGB324 on Slfn11 expression in these tumors appears to be long-lasting, as the protein level stays low 72 hours after treatment. However, there appear to be individual differences, and more than two parallel samples of each time point need to be examined in order to better understand the dynamics of Slfn11 reduction in MV4-11 xenografts after BGB324-treatment.

Example 6: Slfn11 Expression is Reduced in AML Cell Lines after BGB324 Treatment

[0235] Microarray and western blot results from MV4-11 xenografts show downregulation of SLFN11 after BGB324 treatment. This led to the investigation of whether other AML cell lines also exhibited Slfn11 reduction after BGB-324 treatment in vitro.

[0236] MOLM-13, OCI-M1 and OCI-AML3 cells were seeded at different cell densities (corresponding to length of treatment) and treated with BGB324 at IC50-values for 24, 48 and 72 hours. By flow cytometry analysis it was shown that treatment with BGB24 resulted in a marked reduction of SLFN11 expression at nearly all time points in these cells (FIG. 8).

[0237] Next, the dose-dependence of the reduction in Slfn11 expression on BGB324 treatment was investigated.

[0238] MOLM-13 cells treated with increasing doses of BGB324 for 48 hours showed a dose-dependent reduction in Slfn11 expression by western blotting (FIG. 8A). These results were confirmed by flow cytometry (FIG. 9B) using the same antibody.

Example 7: Total Protein Expression of SLFN11 in the AML Cell Panel after Long-Term Treatment (24, 48 and 72 Hours) with BGB324

[0239] When examining total protein expression of SLFN11 in the AML cell panel after long-term treatment (24, 48 and 72 hours) with BGB324, a significant reduction was found at all time points in MOLM13 and Mv4-11 at the highest treatment dose (0.3 μM) (FIG. 10).

[0240] In Mv4-11, SLFN11 was also significantly reduced at treatment with 0.1 uM BGB324 for 72 hours. In Kasumi and OCI-AMLS, the opposite was found; a significant increase of SLFN11 expression, but only after 72 hours of treatment. At 24 and 48 hours, there was no significant differences between treated and control cells.

[0241] Thus, SLFN11 is oppositely regulated in the ‘responding’ and the ‘non-responding’ cells, indicating that a reduction of SLFN11 indicates a biological response to BGB324.

Example 8: Screening of Selected Biomarkers in In Vivo Samples from a MOLM13 Systemic Xenograft Model

[0242] Material from an in vivo MOLM13 systemic model was also evaluated by flow cytometry. Expression of the previously evaluated biomarkers was examined in cells isolated from bone marrows and spleens of animals with systemic AML disease (MOLM13, inoculated for 7 days prior to treatment) treated with BGB324 at 50 mg/kg for 4 days.

[0243] Cells harvested from spleens, blood and bone marrows of the animals were stained with anti CD33 and CD45 antibodies, to determine if systemic disease was established. CD33/CD45-positive cells were identified in spleens (around 10-15%), bone marrows (35-40%) and in the blood (2-6%) of the animals, confirming that the disease was established. There were no significant differences in the percentage of leukemic cells in the BGB324-treated vs. vehicle-treated mice in spleens or bone marrows, but there were a significantly higher percentage of leukemic cells in the blood of BGB324-treated mice (FIG. 11A).

[0244] Cells isolated from spleens and bone marrows were assessed for phosphorylation of Erk, PLCγ1 and Akt, and expression of PHGDH and SLFN11. The samples were also co-stained with CD33, and biomarker expression was only evaluated in CD33-positive cells. A significant reduction of pErk, pPLCγ1, PHGDH and SLFN11 in the bone marrows was observed, and significant reduction of pErk, pPLCγ1 and PHGDH in the spleens (FIG. 22B). pAkt when down in both tissues after treatment, but due to a large standard deviation in the control group, this change was not significant.

INDUSTRIAL APPLICATION

[0245] The invention is industrially applicable through operation of methods in accordance with the invention.