METHODS FOR THE TREATMENT OF CANCERS THAT HAVE ACQUIRED RESISTANCE TO KINASE INHIBITORS

Abstract

Resistance to kinase inhibitors exemplifies the greatest hindrance to effective treatment of cancer patients. Recent studies have suggested that the onset of said resistance might not only be explained by a drug selection of pre-existing resistant sub-clones as it what was generally assumed, but may also arise de novo from a small population of drug-tolerant cells (DTC) that initially resists the treatment by entering a slow cycling state. Thus, targeting these DTC should be a new promising approach to hamper the emergence of secondary resistance to kinase inhibitors. The inventors now demonstrate that farnesyltransferase (but not geranylgeranyl transferase) inhibition can prevent the emergence of said resistance in different oncogenic contexts. In particular, the inventors determined invitro the efficacy of farnesyltransferase inhibitor (i.e. Tipifarnib) in combination with erlotinib in several EGFR-mutated cell lines. They showed that the combination efficiently eliminated all drug tolerant cells, and fully prevented the emergence of resistant clones. Interestingly, similar results were observed in other oncogenic models such as ALK-translocated lung cancer cells or BRAF-mutated melanoma cells. Thus the present invention relates to use of farnesyl transferase inhibitors for the treatment of cancers that have acquired resistance to kinase inhibitors.

Claims

1. A method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective combination comprising a kinase inhibitor and a farnesyltransferase inhibitor.

2. A method delaying and/or preventing development of a cancer resistant to a kinase inhibitor in a subject comprising administering to the subject a therapeutically effective amount of the kinase inhibitor in combination with a farnesyltransferase inhibitor.

3. (canceled)

4. A method of preventing or treating resistance to an administered kinase inhibitor in a subject suffering from a cancer comprising administering to the subject a therapeutically effective amount of a farnesyltransferase inhibitor.

5. (canceled)

6. The method according to claim 1, wherein the kinase inhibitor is an inhibitor targeting one or more targets selected from the group consisting of an EGFR, ALK, B-Raf, MEK, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, IGF1R, c-Met, JAK family, PDGFR α and β, RET, AXL, c-KIT, TrkA, TrkB, TrkC, ROS1, BTK and Syk.

7. The method of claim 6 wherein the kinase inhibitor is selected from the group consisting of gefitinib, erlotinib, lapatinib, vandetanib, afatinib, osimertinib, neratinib, dacomitinib, brigatinib, canertinib, naquotinib, nazartinib, pelitinib, rociletinib, icotinib, AZD3759, AZ5104 (CAS NX 1421373-98-9), poziotinib, WZ4002, Crizotinib, entrectinib, ceritinib, alectinib, lorlatinib, TSR-011, CEP-37440, ensartinib, Vemurafenib, dabrafenib, regorafenib, PLX4720, Cobimetinib, Trametinib, Binimetinib, Selumetinib, PD-325901, CI-1040, PD035901, U0126, TAK-733, Lenvatinib, Debio-1347, dovitinib, BLU9931, Sorafenib, sunitinib, lestaurtinib, tandutinib, quizartinib, crenolanib, gilteritinib, ponatinib, ibrutinib, Linsitinib, NVP-AEW541, BMS-536924, AG-1024, GSK1838705A, BMS-754807, PQ 401, ZD3463, NT157, Picropodophyllin (PPP), Tivantinib, JNJ-38877605, PF-04217903, foretinib (GSK 1363089), Merestinib, Ruxolitinib, tofacitinib, oclacitinib, baricitinib, filgotinib, cerdulatinib, gandotinib, momelotinib, pacritinib, PF-04965842, upadacitinib, peficitinib, fedratinib, imatinib, pazopanib, Telatinib, bosutinib, nilotinib, cabozantinib, Bemcentinib, amuvatinib, gilteritinib (ASP2215), glesatinib (MGCD 265), SGI-7079, Larotrectinib, RXDX-102, altiratinib, LOXO-195, sitravatinib, TPX-0005, DS-6051b, fostamatinib, entospletinib and TAK-659.

8. The method of claim 6 wherein the kinase inhibitor is selected from the group consisting of a EGFR inhibitor, an ALK inhibitor and a B-Raf inhibitor, and the protein kinase inhibitor is selected from the group consisting of gefitinib, erlotinib, lapatinib, vandetanib, afatinib, osimertinib, neratinib, dacomitinib, brigatinib, canertinib, naquotinib, nazartinib, pelitinib, rociletinib, icotinib, AZD3759, AZ5104 (CAS Ns 1421373-98-9), poziotinib, WZ4002, Crizotinib, entrectinib, ceritinib, alectinib, lorlatinib, TSR-011, CEP-37440, ensartinib, Vemurafenib, dabrafenib, regorafenib and PLX4720.

9. The method of claim 6 wherein the kinase inhibitor is a EGFR inhibitor selected from the group consisting of gefitinib, erlotinib, lapatinib, vandetanib, afatinib, osimertinib, neratinib, dacomitinib, brigatinib, canertinib, naquotinib, nazartinib, pelitinib, rociletinib, icotinib, AZD3759, AZ5104 (CAS Ns 1421373-98-9), poziotinib and WZ4002.

10. The method according to claim 1, wherein the subject suffers from an EGFR-mutated cancer, a ALK-mutated cancer, a RAS-mutated cancer, a Met-mutated cancer or a RAF-mutated cancer.

11. The method of claim 10 wherein the cancer is selected from the group consisting of leukemia, lymphoma, sarcoma, melanoma, and cancers of the head and neck, kidney, ovary, pancreas, prostate, thyroid, lung, esophagus, breast, bladder, brain, colorectum, liver, and cervix.

12. The method of claim 10 wherein the subject suffers from a non-small cell lung cancer.

13. The method of claim 10 wherein if the kinase inhibitor is an EGFR inhibitor, the cancer is selected from the group consisting of lung cancer, pancreatic cancer, breast cancer, thyroid cancer, colorectal cancer, cell carcinoma of the head and neck and glioma.

14. The method of claim 10 wherein if the kinase inhibitor is an ALK inhibitor, and the cancer is non-small cell lung cancer.

15. The method of claim 10 wherein if the kinase inhibitor is a B-Raf inhibitor, the cancer is selected from the group consisting of melanoma, lung cancer, colorectal cancer and gastro-intestinal stromal cancer.

16. The method according to claim 1, wherein the farnesyltransferase inhibitor is tipifarnib.

17. A pharmaceutical composition or a kit (kit-of-parts) comprising a farnesyltransferase inhibitor and a kinase inhibitor.

18. The method of claim 13 wherein the lung cancer is non-small cell lung cancer (NSLC), the breast cancer is early breast cancer, the thyroid cancer is medullary thyroid cancer, and the colorectal cancer is metastatic or advanced colorectal cancer.

19. The method of claim 1, wherein the kinase inhibitor is selected from the group consisting of gefitinib, erlotinib, lapatinib, vandetanib, afatinib, osimertinib, neratinib, dacomitinib, brigatinib, canertinib, naquotinib, nazartinib, pelitinib, rociletinib, icotinib, AZD3759, AZ5104 (CAS Ns 1421373-98-9), poziotinib, WZ4002, Crizotinib, entrectinib, ceritinib, alectinib, lorlatinib, TSR-011, CEP-37440, ensartinib, Vemurafenib, dabrafenib, regorafenib, PLX4720, Cobimetinib, Trametinib, Binimetinib, Selumetinib, PD-325901, CI-1040, PD035901, U0126, TAK-733, Lenvatinib, Debio-1347, dovitinib, BLU9931, Sorafenib, sunitinib, lestaurtinib, tandutinib, quizartinib, crenolanib, gilteritinib, ponatinib, ibrutinib, Linsitinib, NVP-AEW541, BMS-536924, AG-1024, GSK1838705A, BMS-754807, PQ 401, ZD3463, NT157, Picropodophyllin (PPP), Tivantinib, JNJ-38877605, PF-04217903, foretinib (GSK 1363089), Merestinib, Ruxolitinib, tofacitinib, oclacitinib, baricitinib, filgotinib, cerdulatinib, gandotinib, momelotinib, pacritinib, PF-04965842, upadacitinib, peficitinib, fedratinib, imatinib, pazopanib, Telatinib, bosutinib, nilotinib, cabozantinib, Bemcentinib, amuvatinib, gilteritinib (ASP2215), glesatinib (MGCD 265), SGI-7079, Larotrectinib, RXDX-102, altiratinib, LOXO-195, sitravatinib, TPX-0005, DS-6051b, fostamatinib, entospletinib and TAK-659; and the farnesyltransferase inhibitor is selected from the group consisting of tipifarnib, lonafamib, FTI-277, GGTI-298, BMS-214664, L-778 and L-123.

20. The method of claim 19, wherein the kinase inhibitor is osimertinib and the farnesyltransferase inhibitor is tipifarnib.

21. The method of claim 10, wherein the cancer is an ALK-mutated NSLC and the kinase inhibitor is an ALK inhibitor; the cancer is a Met-mutated NSLC and the kinase inhibitor is a Met inhibitor; the cancer is a BRAF-mutated metastatic melanoma and the kinase inhibitor is a BRAF inhibitor and/or a MEK inhibitor; the cancer is a BRAF-mutated NSCLC and the kinase inhibitor is a BRAF inhibitor; the cancer is a BRAF-mutated thyroid cancer the kinase inhibitor is a BRAF inhibitor; or the cancer is a BRAF-mutated colorectal cancer and the kinase inhibitor is a BRAF inhibitor and/or a EGFR inhibitor.

22. The method of claim 1, wherein the cancer is EGFR-mutated NSCLC, the kinase inhibitor is osimertinib and the farnesyltransferase inhibitor is tipifarnib.

Description

FIGURES

[0067] FIG. 1. FTi but not GGTi prevent relapse in several TKI-sensitive models. GFP-transduced EGFR-mutated cell lines were treated with Erlotinib at 1 μM with or without FTi (Tipifarnib, 1 μM), GGTi (GGTi-298, 1 μM) or TatC3 (2 μg/ml) (A-C), or Tipifarnib at 0.1 μM (D-F), and response as well as relapse was followed by fluorescence detection. (G-H). GFP-transduced H3122 (ALK-translocated NSCLC cell line) or A375 (BRAF-mutated melanoma cell line) were treated by Tipifarnib 0.1 μM in combination with Alectinib (2 μM) or Vemurafenib (5 μM), respectively, and response as well as relapse was followed by fluorescence detection.

[0068] FIG. 2. (A) Evolution of tumor volume upon indicated treatments. (B) Evolution of tumor size vs baseline at best response (45 days). (C) Kaplan-Meier progression-free survival plot.. (D) Kaplan-Meier overall survival plot. (E) Evolution of mice body size during treatment.

Example

[0069] In Vitro:

[0070] We recently reported that the RAS-related GTPase RHOB has a pivotal role in preventing cell death through the AKT pathway in EGFR-mutated lung cancer cells treated with EGFR-TKI.sup.18. We found that high RHOB tumor levels predict the early relapse of NSCLC patients harbouring EGFR-activating mutations treated with EGFR-TKI. This was also true in BRAF-mutated melanomas treated with the BRAF inhibitor vemurafenib.sup.19, suggesting that the RHOB pathway could be a common adaptive mechanism to receptor tyrosine kinase (RTK)—ERK pathway inhibition that might induce the acquisition of a DTC state. We have also identified a new phenotype related to drug tolerance in vitro after EGFR-TKI treatment that shares several characteristics of a known process of Therapy-Induced Senescence (TIS).sup.20 but also displays some specific features (data not shown). We will thus refer this phenotype to as “senescent-like”. These observations arise from an extensive phenotypic characterization of the DTC state in a panel of EGFR-mutated lung cell lines (that were previously cloned to avoid the presence of potential resistant sub-clones in the bulk population) including the well described PC9 but also HCC827, HCC4006, H3255, and HCC2935 which all display initial sensitivity to EGFR-TKI but have not been yet characterized for their ability to produce DTC in response to EGFR-TKI. Surprisingly, although all these cell lines were able to generate DTC after several days of EGFR-TKI treatment (erlotinib or osimertinib at 1 μM), we observed a high variability intra- and inter-cell lines for several critical parameters such as cell division rate/cell arrest or kinetics of proliferative clones' onset. For instance, PC9, HCC827 and HCC4006 were able to generate proliferative resistant clones after erlotinib treatment, but we never observed resistant clones after erlotinib treatment in HCC2935 and H3255 cell lines (data not shown).

[0071] Despite these differences, we also observed that a common feature of the DTC state among the cell lines was a cell shape reorganization during treatment, mainly a flattened and enlarged morphology, consistent with a TIS process (data not shown). We further explore these morphological changes and we observed a strong increase in actin stress fibers production a few days after initiation of TKI treatment (data not shown). Actin polymerization is a tightly regulated process orchestrated by GTPases. Given our knowledge on the role of RHOB in resistance to targeted therapy, we assessed whether this GTPase could be responsible for the production of stress fibers in response to EGFR-TKI. We first observed that RHOB protein expression and activity were highly increased in DTC in all cell lines, whereas RHOA and RHOC were strongly inhibited (data not shown). We also found that siRNA-specific inhibition of RHOB as well as pharmacological inhibition of RHO-GTPases using C3 exoenzyme (tatC3) not only strongly decreased the production of actin stress fibers but also strongly decreased DTC survival, suggesting a link between actin remodelling and drug-tolerance (data not shown).

[0072] RHOB has no clinically-compatible specific inhibitor, however its activity is dependent on its prenylation status (either farnesylated or geranylgeranylated) and thus can be targeted by famesyltransferase inhibitors (FTi) or geranylgeranyl transferase inhibitors (GGTi).sup.21-23. Therefore, we decided to determine in vitro the efficacy of FTi or GGTi in combination with erlotinib in several EGFR-mutated cell lines (PC9, HCC827 and HCC4006). Combination with GGTI 298 at 1 μM didn't prevent the emergence of resistant proliferative clones (FIGS. 1A-C), whereas combination with FTi Tipifarnib efficiently eliminated all drug tolerant cells when used at 1 μM (FIGS. 1A-C) but also at 0.1 μM (FIGS. 1D-F), and fully prevented the emergence of resistant clones. Interestingly, similar results were observed in other oncogenic models such as ALK-translocated lung cancer cells (e.g. H3122) treated with Alectinib (FIG. 1G) or BRAF-mutated melanoma cells (A375) treated with Vemurafenib (FIG. 1H), suggesting that co-treatment with Tipifarnib could interfere with other targeted therapies that target (RTK)—ERK pathway.

[0073] Tipifarnib used alone at 0.1 μM showed little-to-non effect on PC9 and HCC827 cells growth (data not shown), but showed some cytostatic effect on HCC4006 (data not shown), A375 and H3122, that was exacerbated when Tipifarnib was used at 1 μM (data not shown). Importantly, combination of Tipifarnib (0.1p m) and Erlotinib (1 μM) resulted in complete cell death revealed by the absence of remaining DTC after several days of treatment (data not shown). Interestingly, same results were observed with third generation EGFR-TKI Osimertinib that will be now used as standard first-line treatment for NSCLC patients harbouring EGFR mutations (data not shown).

[0074] Altogether, our in vitro data strongly suggest that Famesyltransferase (but not geranylgeranyl transferase) inhibition can prevent the emergence of resistances to Tyrosine Kinase Inhibitors in different oncogenic contexts. Excitingly, a recently published phase I clinical trial reported that combination of Erlotinib and Tipifarnib was well tolerated in patients.sup.24, however the efficiency of the combination is not indicative since this study was not performed on EGFR-mutated NSCLC patients.

[0075] In Vivo

[0076] Previously described EGFRL858R/T790M lung Patient Derived Xenograft model (TP103, Pax Ares' lab, CNIO Madrid) was implanted sub-cutaneously in 6-8 week old NSG mice (Charles River) and tumors were allowed to establish, sizes (average 300-350 mm3) were matched and then mice were randomly allocated to the following groups: vehicle (n=3), Tipifarnib (n=3), Osimertinib (n=6) and Osimertinib+Tipifarnib (n=6). Tipifanib was administrated by oral gavage at 80 mg/Kg twice a day, 5 days/week and Osimertinib was administrated by oral gavage at 5 mg/Kg once a day, 5 days/week. Tumor size was determined by caliper measurements of tumor length and width and tumor volume was calculated as volume=0.5236×length×width2 (mm), and the mice were weighed once a week. GraphPad Prism (GraphPad Software) was used to perform unpaired two-tailed t-test or Mantel-Cox for PFS and OS plot (FIGS. 2A to 2E).

REFERENCES

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