Triple therapy inhibiting EGFR, RAF1, and STAT3 for pancreatic ductal adenocarcinoma

Abstract

The present invention relates to pharmaceutical compositions comprising inhibitors of c-Raf expression, activity and / or function, inhibitors of EGF receptor (EGFR) expression, activity and / or function, and inhibitors of STAT3 expression, activity and / or function, and their use in the treatment of pancreatic cancer.

Description

[Technical Field]

[0001] The present invention relates to combination cancer therapies that have proven particularly effective in the prevention and treatment of pancreatic cancer. [Background technology]

[0002] Pancreatic cancer is one of the most lethal cancers, with a nearly equal number of deaths and cases, and it is predicted to become the second most deadly cancer by 2030. The 5-year survival rate is only 5%, with a median survival time of less than 6 months. However, minimal improvements have been made in the field of treatment. Currently, surgical resection remains the only curative treatment option for early-stage localized disease, representing 20–30% of patients. Unfortunately, due to late diagnosis, most patients do not benefit from surgery because, at the time of diagnosis, they present with advanced disease with metastasis. Cytotoxic chemotherapy is the standard of care, with overall survival ranging from weeks to months (Nevala-Plagemann et al., 2020). Currently, no targeted therapies are available for patients with pancreatic ductal adenocarcinoma (PDAC). To date, adjuvant therapy with 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX) has been demonstrated to provide the longest median overall survival (OS) (54 months) in patients with resectable disease (Conroy et al., 2018). For patients with advanced or metastatic disease, standard treatment consists of combinations of modern highly cytotoxic agents, such as nab-paclitaxel and gemcitabine or FOLFIRINOX, but these only provide modest improvements in OS, ranging from weeks to months (Conroy et al., 2018; Von Hoff et al., 2013). Undoubtedly, there is an urgent need to develop novel targeted therapies that directly block specific oncogenic pathways with low toxicity. Indeed, a recent consensus statement from the National Cancer Institute (NCI) indicated the need for targeted agents, predictive biomarkers, and improved preclinical models for PDAC (Philip et al., 2009).

[0003] Few preclinical studies have published on the combination of EGFR or STAT3 inhibition with other compounds. In 2011, Nagaraj et al. reported that only the combination of dasatinib and erlotinib with gemcitabine overcame STAT3-dependent resistance to EGFR and Src inhibition both in vitro and in vivo. The pharmacological combination used in this study included only the EGFR inhibitor erlotinib (Nagaraj et al., 2011). In 2012, Navas et al. demonstrated that inhibition of EGFR signaling with erlotinib alone was not sufficient to target pancreatic cancer. Furthermore, the combination of a phosphatidylinositol 3-kinase (PI3K) inhibitor with Stat3 shRNA (short hairpin RNA or small hairpin RNA (ribonucleic acid)) potently inhibited the growth of all tumor cell lines (Navas et al., 2012). In 2015, Zhou et al. demonstrated that STAT3 was significantly activated after MEK inhibition using AZD6244, PD98059, and trametinib in Kras-mutant pancreatic and colorectal cancer cells. Consequently, dual inhibition of STAT3 and MEK exerted significant antitumor effects in Kras-mutant pancreatic cancer cells in vitro (Zhao et al., 2015). In 2017, Sahu et al. reported that MAP2K (MEK) inhibitors were most effective in targeting PDAC spheroids, while combination with the multikinase inhibitor ponatinib was effective in targeting pancreatic cancer cells in both monolayer and spheroid forms. They also demonstrated that combined treatment with an MEK inhibitor and ponatinib resulted in significant tumor regression in a xenograft model. PDAC patient samples also provided evidence of increased STAT3 activation in PDAC tumors and MAPK1 (ERK) activation in liver metastases, implicating STAT3 and ERK as key drivers in primary tumors and metastases, respectively ( Sahu et al., 2017 ).In 2019, Blasco et al. demonstrated that genetic ablation of Egfr and Raf1 resulted in complete regression of a significant portion of small-sized tumors driven by Kras / Trp53. However, some small tumors and most of the larger-sized tumors remained refractory to this combined deletion (Blasco et al., 2019 and WO 2020020942(A1)). DETAILED DESCRIPTION OF THE INVENTION

[0004] The present inventors have demonstrated that simultaneous inhibition of the expression, activity, and / or function of EGFR, RAF1, and STAT3 in PDAC results in significant therapeutic effects with extremely low toxicity.Previous studies on dual therapy against EGFR and RAF1 have shown that this therapy is ineffective in larger, more aggressive PDAC, which do not respond to dual therapy.However, these larger, more aggressive PDAC can be effectively targeted by triple therapy.The present invention is an improved therapy for the treatment of pancreatic cancer because it results in complete tumor regression while its side effects are far less than those of other therapies previously described.

[0005] In a first aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: inhibitors of ac-Raf expression, activity and / or function; b. Inhibitors of EGF receptor (EGFR) expression, activity and / or function; c. Inhibitors of STAT3 expression, activity and / or function The present invention relates to a pharmaceutical composition comprising:

[0006] In a preferred embodiment of the first aspect of the present invention, the inhibitor of the expression, activity and / or function of each one of c-Raf, EGFR or STAT3 comprises at least an inhibitor selected from an inhibitory compound, an inhibitory antibody or antigen-binding fragment thereof, a peptide, a nucleotide sequence, a proteolysis targeted chimera (PROTAC), and any combination thereof.

[0007] In a preferred embodiment of the first aspect of the invention, when the inhibitor of c-Raf, EGFR, or STAT3 expression, activity and / or function comprises a nucleotide sequence, the nucleotide sequence is or encodes a guide RNA, an interfering RNA (e.g., a short hairpin RNA or small hairpin RNA (shRNA) or a microRNA).

[0008] In a preferred embodiment of the first aspect of the present invention, the inhibitor of c-Raf expression, activity and / or function does not inhibit b-Raf expression, activity and / or function. In a preferred embodiment of the first aspect of the present invention, the inhibitor of c-Raf expression, activity and / or function does not inhibit C-RAF kinase activity. In a preferred embodiment, the inhibitor of c-Raf expression, activity and / or function inhibits c-Raf expression or promotes C-RAF degradation. In another preferred embodiment, the inhibitor of c-Raf expression, activity and / or function blocks C-RAF kinase-independent activity.

[0009] In a preferred embodiment, the present invention relates to a pharmaceutical composition comprising an inhibitor of c-Raf expression, activity and / or function and an inhibitor of EGF receptor (EGFR) expression, activity and / or function, wherein the inhibitor of c-Raf expression, activity and / or function is (i) does not inhibit C-RAF kinase activity; (ii) inhibiting c-Raf expression; (iii) promotes the degradation of C-RAF; and / or (iv) inhibits C-RAF kinase-independent activity.

[0010] In a preferred embodiment of the first aspect of the present invention, the inhibitor of c-Raf expression, activity and / or function is other than sorafenib, vemurafenib, dabrafenib and LY3009120. In a preferred embodiment, the inhibitor of c-Raf expression, activity and / or function promotes the degradation of C-RAF. In a more preferred embodiment, the inhibitor is a proteolysis-targeted chimera (PROTAC).

[0011] In a preferred embodiment of the first aspect of the present invention, the inhibitor of EGFR expression, activity and / or function is selected from afatinib, erlotinib, gefitinib, brigatinib, icotinib, neratinib, lapatinib, vandetanib, osimertinib, cetuximab, panitumumab, necitumumab, nimotuzumab, zalutumumab, matuzumab, and combinations thereof.

[0012] In a preferred embodiment of the first aspect of the present invention, the inhibitor of STAT3 expression, activity and / or function is an shRNA or a PROTAC.

[0013] In a preferred embodiment of the first aspect of the present invention, the composition comprises a therapeutically effective amount of an inhibitor of c-Raf expression, activity and / or function, a therapeutically effective amount of an inhibitor of EGFR expression, activity and / or function, a therapeutically effective amount of an inhibitor of STAT3 expression, activity and / or function, and optionally a pharmaceutically acceptable excipient.

[0014] In another aspect, the present invention relates to the pharmaceutical composition of the first aspect for use in the prevention and / or treatment of cancer. Preferably, it is for use in the prevention and / or treatment of pancreatic cancer. The present invention also relates to the pharmaceutical composition of the first aspect for use in the prevention and / or treatment of pancreatic intraepithelial neoplasia or pancreatic ductal adenocarcinoma. Preferably, the present invention relates to the pharmaceutical composition of the first aspect for use in tumor regression in subjects suffering from the above cancers.

[0015] The present invention also relates to the pharmaceutical composition of the first aspect for use in tumor regression in a subject afflicted with pancreatic ductal adenocarcinoma (PDAC).The present invention also relates to the pharmaceutical composition of the first aspect for use in tumor regression in a subject who does not respond to a composition comprising only a combination of an inhibitor of c-Raf expression, activity and / or function and an inhibitor of EGFR expression, activity and / or function.

[0016] One aspect of the present invention is a method for preventing and / or treating cancer, comprising administering a therapeutically effective amount of an inhibitor of the expression, activity and / or function of c-Raf, a therapeutically effective amount of an inhibitor of the expression, activity and / or function of EGF receptor (EGFR), and a therapeutically effective amount of an inhibitor of the expression, activity and / or function of STAT3, and optionally a pharmaceutically acceptable excipient.Preferably, the method of the present invention is for treating and / or preventing pancreatic cancer, more preferably pancreatic intraepithelial neoplasia (PanIN) or PDAC. [Brief explanation of the drawings]

[0017] [Figure 1] A. Colony formation assay of Egfrlox / lox;Raf1lox / lox PDAC cells infected with adeno-GFP and adeno-CRE viral particles in combination with small hairpin RNA (shRNA) against STAT3 and Scr (empty vector) as a control. Five thousand cells were seeded for 10 days. B. Western blot analysis of EGFR, RAF1, and STAT3 expression in cell lysate extracts. GAPDH was used as a loading control. [Figure 2] A. Colony formation assay of Egfrlox / lox;Raf1lox / lox PDAC cells infected with adeno-GFP and adeno-CRE viral particles in combination with treatment with 1 μM SD-36. Five thousand cells were seeded for 10 days, and the culture medium was changed daily. B. Western blot analysis of STAT3 expression in cell lysate extracts. GAPDH was used as a loading control. [Figure 3]A. Colony formation assay of Egfrlox / lox, Raf1lox / lox, and Stat3lox / lox PDAC cells infected with adeno-GFP and adeno-CRE viral particles. Five thousand cells were plated for 10 days. B. Western blot analysis of EGFR, RAF1, and STAT3 expression in cell lysate extracts. GAPDH was used as a loading control. [Figure 4] A. Colony formation assay of Egfrlox / lox, Raf1lox / lox, and Stat3lox / lox PDAC cells (T7-T12 cell line) infected with shRNAs against EGFR and RAF1, and Scr (empty vector) as a control. Five thousand cells were seeded for 10 days. B. Western blot analysis of EGFR and RAF1 expression in cell lysate extracts. GAPDH was used as a loading control. [Figure 5] Tumor growth monitored by ultrasound in genetically engineered mice with the genotype (Elas-tTa / tetO-Flp, K-Ras+ / FSFG12V, p53frt / frt, Raf1lox / lox, Rosa26_CreERT2+ / KI) treated with tamoxifen and afatinib (NR doublet) and tamoxifen, afatinib, and SD-36 (NR triplet). [Figure 6] Tumor growth monitored by ultrasound after implantation of three Egfrlox / lox, Raf1lox / lox, and Stat3lox / lox PDAC cell lines (T7-T9) in triplicate. NT: untreated mice before and after cell implantation. TMX: tamoxifen-treated mice, fed a tamoxifen diet from 1 week before cell implantation until the end of the experiment. NT mice were humanely sacrificed due to tumor burden at the last time point shown on the graph. [Figure 7] Tumor growth monitored by ultrasound in mice implanted with three Egfrlox / lox, Raf1lox / lox, and Stat3lox / lox PDAC cell lines (T7–T9) during the tamoxifen diet period.

[0018] Example The present invention is illustrated and explained by the following examples, which are not intended to limit the invention in any way.

[0019] material and method Silencing of Egfr, Raf1, and Stat3 was mediated by lentiviral short hairpin RNA (shRNA) plasmids TRCN0000055218, TRCN0000012628, and TRCN0000071453, respectively. A non-targeting shRNA vector was used as a control. Pharmacological treatment was performed using the following agents: afatinib (20 mg / kg daily), SD-36 (50 mg / kg daily).

[0020] Triple loss of EGFR, RAF1, and Stat3 eliminated all PDAC. Combined ablation of EGFR and RAF1 expression resulted in complete regression of a significant proportion (approximately 50%) of PDAC tumors (response, R, tumors) driven by Kras / Trp53 mutations in genetically engineered mice (Blasco et al., Cancer Cell 2019). Experiments were performed on tumors mutated for the oncogene Kras and the tumor suppressor gene p53 (abbreviated as KPeFC in the figure). Essentially, cases of pancreatic ductal adenocarcinoma have two driving factors: tumor-associated Kras mutations and p53 mutations, both in humans and in mouse models. In a study by Blasco et al., 50% of murine PDAC (non-response, NR, tumors) were insensitive to this therapeutic strategy. Remarkably, triple ablation of EGFR, RAF1, and Stat3 eliminated all PDAC:R and NR tumors.

[0021] STAT3 inhibition (genetic or pharmacological) resulted in cell death of NR tumor cells. STAT3 expression was targeted by shRNA after gene deletion of Egfr and Raf1 in NR cells by adeno-GFP and adeno-GFP-CRE infection. Importantly, this combined therapeutic strategy resulted in cell death of NR tumor cells. Notably, downregulation of Stat3 did not affect NR cells expressing Egfr and Raf1 (Figure 1A, Figure 1B). These results were further validated by a pharmacological approach using the STAT3 PROTAC degrader SD-36 (Figure 2A, Figure 2B). NR tumor cells pre-infected with adeno-GFP and adeno-GFP-CRE viruses to deplete EGFR and Raf1 (floxed targets) were treated with SD-36 for 10 days in a colony formation assay. Target depletion was highly efficient. SD-36 effectively degraded STAT3 without toxic side effects, and NR cells expressing Egfr and Raf1 grew similarly to vehicle controls.

[0022] In vitro triple deletion in non-responsive cells A genetic approach was also performed: triple deletion of Egfr, Raf1, and Stat3. For this, three floxed alleles: Egfr lox / lox ;Raf1 lox / lox ;Stat3 lox / lox We generated a PDAC mouse model bearing EGFR and Raf1. Twenty-five PDAC cell lines were generated for in vitro studies. Simultaneous ablation of the three floxed targets by adeno-CRE infection resulted in complete death of the 25 cell lines. Figures 3A and 3B show examples of six cell lines. Interestingly, the few small colonies that remained were mostly apoptotic and could not be collected for further study. These cell lines were identified as NR based on their response to shRNA-mediated ablation of EGFR and Raf1 (Figures 4A and 4B). Twelve of the 25 cell lines were NR. Thus, ablation of the three targets efficiently induced cell death in NR cells.

[0023] Triple deletion in vivo The potential therapeutic value of simultaneous ablation of Egfr, Raf1, and Stat3 in vivo was also investigated. We introduced three KPeFCs, Egfr, Raf1, and Stat3 into the pancreas of immunocompetent mice (C57BL / 6). lox / lox , Raf1 lox / lox , Stat3 lox / lox Orthotopic studies were performed by inoculating tumor cell lines. These cell lines are NR for Egfr and Raf1 loss (T7-T9, Figure 4A, Figure 4B) and carry the Rosa26CreERT2 allele. Upon exposure to tamoxifen, CreERT2 recombinase is activated, leading to the loss of the floxed allele in tumor cells. To study the effects of triple loss, we performed two different protocols of tamoxifen treatment (added to the diet): immediately after implantation and in established tumors. In the first protocol, tamoxifen treatment was initiated one week before the day of cell implantation into the pancreatic parenchyma. This approach achieved very early target loss (orthotopic initiation study) before tumor detection. Ultrasound monitoring of tumor growth showed that tumors developed in untreated mice within 30 days, whereas mice on the tamoxifen diet did not develop tumors over the study period (100 days) (Figure 5). Therefore, the loss of the three targets in vivo resulted in tumor cell death. As a more therapeutic approach, an orthotopic treatment study was performed in which tamoxifen was administered to tumor-bearing mice. Surprisingly, tumors of different sizes (10–80 mm) were observed after a treatment period of approximately 2 weeks. 3 ) tumor regression was observed. To confirm that the triple elimination of neoplastic cells caused complete and sustained tumor regression, tumor monitoring continued for an additional 35 days under tamoxifen treatment (Figure 6). Thus, simultaneous elimination of the three targets in vivo resulted in complete tumor regression.

[0024] Treatment with tamoxifen genetically abolished Raf1 gene expression. This was also included in the combined treatment of afatinib (a tyrosine receptor kinase inhibitor for EGFR) and SD-36 (a PROTAC degrader for STAT3). Interestingly, dual inhibition of EGFR and RAF1 with afatinib and tamoxifen did not cause any delay in tumor growth in large tumors (NR doublet). However, additional treatment with SD-36 (NR triplet) resulted in a significant delay and slight reduction in tumor growth, demonstrating the therapeutic effect of triple inhibition in NR PDAC compared with the doublet combination.

[0025] References Blasco,TMet al.(2019).Cancer Cell 35,573-587. Conroy, T. et al. (2018). N. Engl. J. Med. 379, 2395-2406. Navas, C. et al. (2012). Cancer Cell. 22, 318-30. Nagaraj, NSet al. (2011). Clin Cancer Res. 17, 483-93. Nevala-Plagemann, C. et al. (2020). Nat. Rev. Clin. Oncol. 2, 108-123. Philip,PAet al.(2009).J Clin Oncol.27,5660-9. Sahu, N. et al. (2017).Mol Cancer Ther.9,1729-1738. Von Hoff,DDet al.(2013).N Engl J Med.369,1691-703. Zhao, C. et al. (2015). Oncotarget 6, 14472-87.

Claims

1. a. an inhibitor of c-Raf expression, activity and / or function; b. an inhibitor of EGF receptor (EGFR) expression, activity and / or function; c. Inhibitors of STAT3 expression, activity and / or function 10. A pharmaceutical composition comprising:

2. 2. The pharmaceutical composition of claim 1, wherein the inhibitor of the expression, activity and / or function of each one of c-Raf, EGFR or STAT3 comprises at least an inhibitor selected from an inhibitory compound, an inhibitory antibody or antigen-binding fragment thereof, a peptide, a nucleotide sequence, a proteolytic targeting chimera (PROTAC), and any combination thereof.

3. 3. The pharmaceutical composition of claim 2, wherein the nucleotide sequence is or encodes a guide RNA, an interfering RNA, an shRNA, or a microRNA.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the inhibitor of c-Raf expression, activity and / or function is other than sorafenib, vemurafenib, dabrafenib and LY3009120.

5. 5. The pharmaceutical composition of any one of claims 1 to 4, wherein the inhibitor of c-Raf expression, activity and / or function does not inhibit b-Raf expression, activity and / or function; and / or the inhibitor of c-Raf expression, activity and / or function does not inhibit C-RAF kinase activity.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the inhibitor of EGFR expression, activity and / or function is selected from afatinib, erlotinib, gefitinib, brigatinib, icotinib, neratinib, lapatinib, vandetanib, osimertinib, cetuximab, panitumumab, necitumumab, nimotuzumab, zalutumumab, matuzumab, and combinations thereof.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the inhibitor of STAT3 expression, activity and / or function is an shRNA or a PROTAC.

8. 8. The pharmaceutical composition according to any one of claims 1 to 7, comprising a therapeutically effective amount of the inhibitor of c-Raf expression, activity and / or function, a therapeutically effective amount of the inhibitor of EGFR expression, activity and / or function, a therapeutically effective amount of the inhibitor of STAT3 expression, activity and / or function, and optionally a pharmaceutically acceptable excipient.

9. A pharmaceutical composition according to any one of claims 1 to 8 for use in the prevention and / or treatment of cancer.

10. The pharmaceutical composition according to any one of claims 1 to 8 for use in the prevention and / or treatment of pancreatic cancer.

11. A pharmaceutical composition according to any one of claims 1 to 8 for use in the prevention and / or treatment of pancreatic intraepithelial neoplasia or of pancreatic ductal adenocarcinoma.

12. A pharmaceutical composition according to any one of claims 9 to 11 for use in tumor regression in a subject suffering from said cancer.

13. A pharmaceutical composition according to any one of claims 9 to 11 for use in tumor regression in a subject afflicted with pancreatic ductal adenocarcinoma (PDAC).

14. 14. The pharmaceutical composition of any one of claims 9 to 13 for use in tumor regression in a subject that does not respond to a composition comprising only a combination of said inhibitor of c-Raf expression, activity and / or function and said inhibitor of EGFR expression, activity and / or function.

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