BRAF-mutant Cells Research Articles

KZ02 enhances the radiosensitivity of BRAF-mutated CRC in vitro and in vivo

Colorectal cancer (CRC) is a common malignant tumor with a high incidence and mortality worldwide. Preoperative chemoradiotherapy is a common treatment for patients with metastatic colorectal cancer (mCRC) as it reduces colostomy and local recurrence. The RAS (rat sarcoma)-RAF (extracellular signal-regulated kinase)-MEK (mitogen-activated protein kinase)-ERK (extracellular signal-regulated kinase) pathway regulates important cellular processes in the CRC. Abnormal ERK activation stimulates cell growth and provides a survival advantage. Our group has previously reported that the compound KZ02 has a stronger ability to inhibit tumor growth than AZD6244 (a MEK inhibitor). In this study, we evaluated the antitumor activity of KZ02 in combination with ionizing radiation (IR) and investigated its mechanism of action in BRAF-mutated colorectal cancer. Our results showed that this combination kills tumor cells better than either radiation or drugs alone, both in vivo and in vitro. Furthermore, studies have shown that KZ02 inhibits ERK overactivation. The combination resulted in a G1 phase arrest, a reduction in the radioresistant S phase, and aggravating DNA damage. It can also inhibit Pim-1 (Moloney murine leukemia virus-1), p-BAD (Bcl-2 associated agonist of cell death), Bcl-2 (B-cell lymphoma 2) and Bcl-XL (B-cell lymphoma-extra large) levels and promote apoptosis when combined with radiation. Our results suggest that KZ02 significantly increases the radiosensitivity of BRAF-mutated CRC cells by perturbing the cell cycle, increasing DNA damage, and promoting tumor apoptosis.

Kisspeptin-mediated improvement of sensitivity to BRAF inhibitors in vemurafenib-resistant melanoma cells.

Metastatic dissemination is still one of the major causes of death of melanoma's patients. KiSS1 is a metastasis suppressor originally identified in melanoma cells, known to play an important physiological role in mammals' development and puberty. It has been previously shown that expression of KiSS1 could be increased in lung cancer cells using epigenetic agents, and that KiSS1 could have a pro-apoptotic action in combination with cisplatin. Thus, the aim of the present study was to examine in human melanoma vemurafenib sensitive- and -resistant BRAF mutant cells characterized by different mutational profiles and KiSS1, KiSS1 receptor and KiSS1 drug-induced release, if peptides derived from KiSS1 cleavage, i.e., kisspeptin 54, could increase the sensitivity to vemurafenib of human melanoma, using cellular, molecular and biochemical approaches. We found that kisspeptin 54 increases vemurafenib pro-apoptotic activity in a statistically significant manner, also in drug resistant cellular models. The efficacy of the combination appears to reflect the intrinsic susceptibility of each cell line to PLX4032-induced apoptosis, together with the different mutational profile as well as perturbation of proteins regulating the apoptotic pathway, The results presented here highlight the possibility to exploit KiSS1 to modulate the apoptotic response to therapeutically relevant agents, suggesting a multitasking function of this metastasis suppressor.

STAG2 inactivation reprograms glutamine metabolism of BRAF-mutant thyroid cancer cells

STAG2, an important subunit in cohesion complex, is involved in the segregation of chromosomes during the late mitosis and the formation of sister chromatids. Mutational inactivation of STAG2 is a major cause of the resistance of BRAF-mutant melanomas to BRAF/MEK inhibitors. In the present study, we found that STAG2 was frequently down-regulated in thyroid cancers compared with control subjects. By a series of in vitro and in vivo studies, we demonstrated that STAG2 knockdown virtually had no effect on malignant phenotypes of BRAF-mutant thyroid cancer cells such as cell proliferation, colony formation and tumorigenic ability in nude mice compared with the control. In addition, unlike melanoma, STAG2 knockdown also did not affect the sensitivity of these cells to MEK inhibitor. However, we surprisingly found that STAG2-knockdown cells exhibited more sensitive to glutamine deprivation or glutaminase inhibitor BPTES compared with control cells. Mechanistically, knocking down STAG2 in BRAF-mutant thyroid cancer cells decreases the protein stability of c-Myc via the ERK/AKT/GSK3β feedback pathway, thereby impairing glutamine metabolism of thyroid cancer cells by down-regulating its downstream targets such as SCL1A5, GLS and GLS2. Our data, taken together, demonstrate that STAG2 inactivation reprograms glutamine metabolism of BRAF-mutant thyroid cancer cells, thereby improving their cellular response to glutaminase inhibitor. This study will provide a potential therapeutic strategy for BRAF-mutant thyroid cancers.

Pin1 inhibitor API-1 sensitizes BRAF-mutant thyroid cancers to BRAF inhibitors by attenuating HER3-mediated feedback activation of MAPK/ERK and PI3K/AKT pathways

BRAFV600E mutation is one of the most therapeutic targets in thyroid cancers. However, its specific inhibitors have shown little clinical benefit because they can reactivate the MAPK/ERK and PI3K/AKT pathways by feedback upregulating the transcription of HER3. Peptidyl-prolyl cis/trans isomerase Pin1 has been proven to be closely associated with tumor progression. Here, we aimed to determine antitumor activity of Pin1 inhibitor API-1 in thyroid cancer and its effect on cellular response to BRAF inhibitors. The results showed that API-1 exhibited strong antitumor activity against thyroid cancer. Meanwhile, it improved the response of BRAF-mutant thyroid cancer cells to BRAF inhibitor PLX4032 and there was a synergistic effect between them. Specially, a combination therapy of API-1 and PLX4032 significantly inhibited cell proliferation, colony formation, and the growth of xenograft tumors as well as induced cell apoptosis in BRAF-mutant thyroid cancer cells compared with API-1 or PLX4032 monotherapy. Similar results were also observed in transgenic mice with BrafV600E-driven thyroid cancer. Mechanistically, API-1 enhanced XPO5 ability to export pre-microRNA 20a (pre-miR-20a) from the nucleus to cytoplasm, thereby promoting the maturation of miR-20a-5p. Further studies showed that miR-20a-5p specifically targeted and down-regulated HER3, thereby blocking the reactivation of MAPK/ERK and PI3K/AKT signaling pathways caused by PLX4032. These results, taken together, demonstrate that Pin1 inhibitor API-1 significantly improves the sensitivity of BRAF-mutant thyroid cancer cells to PLX4032. Thus, this study not only determines the potential antitumor activity of Pin1 inhibitor API-1 in thyroid cancer but also offers an alternative therapeutic strategy for BRAF-mutant thyroid cancers by a combination of Pin1 inhibitor and BRAF kinase inhibitor.

Oncogenic BRAF and p53 Interplay in Melanoma Cells and the Effects of the HDAC Inhibitor ITF2357 (Givinostat).

Oncogenic BRAF mutations have been widely described in melanomas and promote tumour progression and chemoresistance. We previously provided evidence that the HDAC inhibitor ITF2357 (Givinostat) targets oncogenic BRAF in SK-MEL-28 and A375 melanoma cells. Here, we show that oncogenic BRAF localises to the nucleus of these cells, and the compound decreases BRAF levels in both the nuclear and cytosolic compartments. Although mutations in the tumour suppressor p53 gene are not equally frequent in melanomas compared to BRAF, the functional impairment of the p53 pathway may also contribute to melanoma development and aggressiveness. To understand whether oncogenic BRAF and p53 may cooperate, a possible interplay was considered in the two cell lines displaying a different p53 status, being p53 mutated into an oncogenic form in SK-MEL-28 and wild-type in A375 cells. Immunoprecipitation revealed that BRAF seems to preferentially interact with oncogenic p53. Interestingly, ITF2357 not only reduced BRAF levels but also oncogenic p53 levels in SK-MEL-28 cells. ITF2357 also targeted BRAF in A375 cells but not wild-type p53, which increased, most likely favouring apoptosis. Silencing experiments confirmed that the response to ITF2357 in BRAF-mutated cells depends on p53 status, thus providing a rationale for melanoma-targeted therapy.

Prognostic and predictive value of metformin in the European Organisation for Research and Treatment of Cancer 1325/KEYNOTE-054 phase III trial of pembrolizumab versus placebo in resected high-risk stage III melanoma

BackgroundMetformin is a commonly prescribed and well-tolerated medication. In laboratory studies, metformin suppresses BRAF wild-type melanoma cells but accelerates the growth of BRAF-mutated cells. This study investigated the prognostic and predictive value of metformin, including with respect to BRAF mutation status, in the European Organisation for Research and Treatment of Cancer 1325/KEYNOTE-054 randomised controlled trial. MethodsPatients with resected high-risk stage IIIA, IIIB, or IIIC melanoma received 200 mg of pembrolizumab (n = 514) or placebo (n = 505) every 3 weeks for twelve months. Pembrolizumab prolonged recurrence-free survival (RFS) and distant metastasis-free survival (DMFS) at approximately 42 months median follow-up (Eggermont et al., TLO, 2021). Multivariable Cox regression was used to estimate associations of metformin with RFS and DMFS. Interaction terms were used to model effect modification by treatment and BRAF mutation. ResultsFifty-four patients (0.5%) used metformin at baseline. Metformin was not significantly associated with RFS (hazard ratio [HR] 0.87, 95% confidence interval [CI] 0.52–1.45) and DMFS (HR 0.82, 95% CI 0.47–1.44). The interaction between metformin and the treatment arm was not significant for either RFS (p = 0.92) or DMFS (p = 0.93). Among patients with mutated BRAF, the association of metformin with RFS (HR 0.70, 95% CI 0.37–1.33) was greater in magnitude though not significantly different to those without mutated BRAF (HR 0.98, 95% CI 0.56–1.69). ConclusionsThere was no significant impact of metformin use on pembrolizumab efficacy in resected high-risk stage III melanoma. However, larger studies or pooled analyses are needed, particularly to explore a possible effect of metformin in BRAF-mutated melanoma.

HAMLET effect on cell death and mitochondrial respiration in colorectal cancer cell lines with KRAS/BRAF mutations

PurposeTreatment of advanced colorectal cancer (CRC) depends on the correct selection of personalized strategies. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a natural proteolipid milk compound that might serve as a novel cancer prevention and therapy candidate. Our purpose was to investigate HAMLET effect on viability, death pathway and mitochondrial bioenergetics of CRC cells with different KRAS/BRAF mutational status in vitro.MethodsWe treated three cell lines (Caco-2, LoVo, WiDr) with HAMLET to evaluate cell metabolic activity and viability, flow cytometry of apoptotic and necrotic cells, pro- and anti-apoptotic genes, and protein expressions. Mitochondrial respiration (oxygen consumption) rate was recorded by high-resolution respirometry system Oxygraph-2 k.ResultsThe HAMLET complex was cytotoxic to all investigated CRC cell lines and this effect is irreversible. Flow cytometry revealed that HAMLET induces necrotic cell death with a slight increase in an apoptotic cell population. WiDr cell metabolism, clonogenicity, necrosis/apoptosis level, and mitochondrial respiration were affected significantly less than other cells.ConclusionHAMLET exhibits irreversible cytotoxicity on human CRC cells in a dose-dependent manner, leading to necrotic cell death and inhibiting the extrinsic apoptosis pathway. BRAF-mutant cell line is more resistant than other type lines. HAMLET decreased mitochondrial respiration and ATP synthesis in CaCo-2 and LoVo cell lines but did not affect WiDr cells’ respiration. Pretreatment of cancer cells with HAMLET has no impact on mitochondrial outer and inner membrane permeability.

Abstract LB320: Discovery and characterization of QTX3034, a potent, selective, and orally bioavailable allosteric KRAS inhibitor

Abstract The RAS family of proto-oncogenes are the most frequently mutated genes in cancer, in which mutations in KRAS account for approximately 25% of all human cancers. RAS oncogenes impair the ability of RAS to convert from its active GTP-bound form into its inactive GDP-bound state leading to the sustained activation of the MAPK signaling pathway and ultimately driving tumorigenesis. Until recently, KRAS was regarded as undruggable, but newly approved covalent inhibitors of KRASG12C have demonstrated clinical benefit in patients harboring this mutation. However, patients with KRASG12C mutation only represent approximately 10% of the KRAS-mutated or amplified cancer population, and multiple RAS-mediated resistance mechanisms have emerged with these covalent compounds. Here we show that QTX3034 is a potent, highly selective, and orally bioavailable mutant KRAS inhibitor. Using the most common KRAS mutant, KRASG12D, QTX3034 demonstrated picomolar binding affinity (0.6 nM) to the inactive form of KRASG12D and inhibited SOS1/2 mediated nucleotide exchange with picomolar potency (0.2 nM). In a NanoBiT cellular target engagement assay, QTX3034 treatment disrupted KRASG12D:RAF1 protein-protein interaction in a concentration-dependent manner with an EC50 of 17 nM in HEK293T cells. Western blot analyses and CTG proliferation assays illustrated that QTX3034 treatment inhibited downstream ERK phosphorylation and cell proliferation in KRASG12D -driven cancer cell lines. Similar inhibitory effects were observed across KRASG12D and KRASG12V mutant cancer cell lines, albeit less potent against the latter. Notably, QTX3034 produced no effect against NRAS or BRAF mutant cells. In vivo, QTX3034 achieved sustained systemic exposure levels required for efficacy following single oral administration in mice, indicating potential durable inhibition of KRAS signaling in vivo. In a KRASG12D -driven xenograft model, oral administration of QTX3034 twice daily (BID) demonstrated dose-dependent anti-tumor efficacy and achieved tumor regression in 100% of tumors. In a KRASG12V -driven xenograft model, oral administration of QTX3034 showed significant tumor growth inhibition, consistent with its activity against KRASG12V in vitro. QTX3034 was well-tolerated in rodent, repeat dose toxicology studies and exhibited significant exposure in the brain. QTX3034 has good solubility, permeability, and moderate protein binding in human plasma. The systemic clearance in rats and dogs was moderate to high, respectively, with good oral bioavailability. QTX3034 exhibited weak CYP inhibition (direct and time-dependent) with a favorable in vitro off-target selectivity/safety profile. Taken together, this preclinical characterization supports the advancement of QTX3034 into IND-enabling studies for KRAS mutant cancers. Citation Format: Yang W. Zhang, Dave Rominger, Elizabeth Donohue Vo, Jillian M. Silva, Yang J. Zhang, Greg Lee, John Micozzi, Ben Reid, Brooke McDonough, Audrey Hospital, Juan I. Luengo, Cameron Pitt, Hong Lin. Discovery and characterization of QTX3034, a potent, selective, and orally bioavailable allosteric KRAS inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB320.

Abstract 539: Furin predicts prognosis and promotes PTC progression and metastasis via MEK-ERK axis

Abstract Furin belongs to the proprotein convertase family and its expression has been documented in various types of cancers, but its role in Thyroid cancer remains unclear. We investigated the expression of Furin in a large cohort of middle eastern PTC patient’s samples and explored its functional role and mechanism in PTC cell line in vitro and in vivo. Furin overexpression was observed in 44.6% of all PTC cases and it was significantly associated with aggressive clinicopathological parameters and poor outcome. We show that inactivation of Furin suppresses tumor growth, proliferation, migration, invasion, spheroid growth and progression of EMT in BRAF mutant cells, whereas its overexpression in BRAF wild-type PTC cell lines reversed the effect. Mechanistically, Furin inactivation caused MEK/ERK pathway suppression in BRAF mutant cells, while inhibition of MEK has no effect on Furin expression which suggest Furin is a functional upstream of MEK/ERK Pathway. Furthermore, study revealed the synergistic antitumor effect of Furin depletion and MEK inhibitor. These results indicate that Furin is an important prognostic marker and its inhibition can be a potential therapeutic target for aggressive PTC. Citation Format: Pratheeshkumar Poyil, Abdul K. Siraj, Divya Padmaja, Sandeep Kumar Parvathareddy, Roxanne Diaz, Saravanan Thangavel, Rafia Begum, Wael Haqawi, Khadija Alobaisi, Falah Hassan Al-Mohanna, Saif S. Al-Sobhi, Fouad Al-Dayel, Khawla S. Al-Kuraya. Furin predicts prognosis and promotes PTC progression and metastasis via MEK-ERK axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 539.

Abstract 4155: The RAF/MEK clamp avutometinib (VS-6766) induces an immunogenic tumor microenvironment and potentiates the efficacy of anti-PD-1

Abstract The RAS/RAF/MEK/ERK (MAPK) pathway is one of the most commonly mutated oncogenic pathways in human cancers. Although RAS, RAF and MEK have been validated as anticancer targets with approval of KRAS G12C, BRAF and MEK inhibitors, combination strategies with chemotherapy, targeted therapies and/or immune checkpoint inhibitors may be optimal for deep and durable response. Avutometinib (avuto, VS-6766) is a unique RAF/MEK clamp that potently inhibits MEK kinase activity and induces a dominant negative RAF/MEK complex preventing phosphorylation of MEK by ARAF, BRAF and CRAF. Preclinically, avuto has shown strong anti-proliferative potency across tumor cell lines carrying various MAPK pathway alterations. Clinically, avuto monotherapy has shown responses in gynecological cancers and KRAS mutant non-small cell lung cancer. Here, we tested the immune modulatory effects of avuto on tumor cells and tumor-infiltrating immune cells and assessed anti-tumor efficacy in mice treated with avuto in combination with an anti-PD1 antibody. In a panel of KRAS and BRAF mutant human tumor cell lines, avuto treatment increased the expression of MHC-I complex genes, including B2M, HLA-A and TAP1. In the CT26 KRAS G12D syngeneic colorectal cancer model, upregulation of B2M and TAP1 by avuto was confirmed in vivo, suggesting that avuto may increase antigen presentation. Furthermore, avuto upregulated the expression of markers of T cells (CD8, PDCD1), NK cell activation (NCR1) and interferon response (IFNG, IRF7, IL12) in the CT26 model. Interestingly, all these pro-immune changes observed with avuto were stronger than those observed with an equivalent dose level of the MEK-only inhibitor trametinib. Avuto also increased expression of B2M, CD8A, PDCD1, NCR1 and IRF7 in a KPARG12C orthotopic lung cancer model. Flow cytometry analysis showed that avuto also significantly increased MHC-II expression by tumor cells and the numbers of CD8 T cells and M1 macrophages, and significantly decreased monocytic and granulocytic MDSCs. These immune changes indicate that avuto induces an immunogenic tumor microenvironment that may potentiate the efficacy of immuno-oncology agents such as anti-PD-1. Accordingly, in the CT26 model, whereas avuto and anti-PD-1 each delayed tumor growth, combination of avuto with anti-PD-1 increased antitumor efficacy and prolonged survival. Furthermore, all complete responders in the avuto + anti-PD-1 group were able to reject a re-challenge with CT26 tumor cells and showed increased CD8 and CD4 effector memory T cells relative to untreated naïve control mice, indicating that avuto + anti-PD-1 treatment induces durable immune memory. These results support clinical evaluation of avutometinib in combination with an anti-PD-1 antibody for treatment of patients with solid tumors harboring MAPK pathway alterations such as KRAS or BRAF mutations. Citation Format: Silvia Coma, Miriam Molina Arcas, Julian Downward, Jonathan A. Pachter. The RAF/MEK clamp avutometinib (VS-6766) induces an immunogenic tumor microenvironment and potentiates the efficacy of anti-PD-1. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4155.

Abstract 3402: In vivo efficacy of RAF/MEK clamp avutometinib (VS-6766) in combination with FAK inhibition in low grade serous ovarian cancer

Abstract Objective: Low grade serous carcinoma (LGSC) of the ovary is characterized by a high recurrence rate, resistance to most chemotherapeutic agents, and limited effective therapeutic options. Recently, LGSC has been found to often contain mutations in KRAS (~30%), NRAS (~10%), and BRAF (~8%). Avutometinib (VS-6766) is a RAF/MEK clamp that induces dominant negative complexes of RAF with MEK, and inhibits proliferation of KRAS, NRAS, BRAF, and CRAF mutant cell lines. VS-4718 is a focal adhesion kinase (FAK) inhibitor that has shown synergistic anti-tumor activity with avutometinib in other cancer models, as it blocks the potential of FAK to function as an adaptive resistance mechanism to RAF/MEK inhibition. This study aimed to determine the efficacy of the RAF/MEK clamp avutometinib with FAK inhibition in vivo in LGSC patient derived tumor xenograft (PDX) models. Methods: Whole-exome sequencing was used to evaluate mutations in two primary ovarian LGSC PDX models (OVA(K)250 and PERIT(M)17). OVA(K)250 and PERIT(M)17 cells were injected into SCID mice and animals were treated with either vehicle control (CTRL), RAF/MEK clamp avutometinib (MEKi), FAK inhibitor VS-4718, or the combination (MEKi/FAKi). Tumor volumes were measured twice per week. Results: Both the OVA(K)250 and PERIT(M)17 PDX models express wildtype KRAS and express RAF1 mutations at position 12641679 A>C, V207G. In PDX OVA(K)250 mice treated with MEKi/FAKi, day 9 tumor volume was significantly lower compared to mice treated with CTRL (p=0.0002). At day 9, tumor volume was also significantly lower in mice treated with MEKi alone compared to CTRL (p=0.0007). Mice treated with FAKi alone had tumor volumes that were significantly smaller than CTRL by day 12 (p=0.009). By day 23, CTRL mice had reached tumor volume characteristics requiring sacrifice. Combination MEKi/FAKi treatment resulted in improved overall survival (OS); median OS for combination or MEKi alone was not reached by 60 days. Treatment with FAKi led to median OS of 35 days; CTRL mice had median OS of 23 days (p=0.001). In vivo experiments with PDX PERIT(M)17 and PDX OVA(K)250 are ongoing. Conclusion: Combination of avutometinib with FAK inhibition showed improved in vivo preclinical anti-tumor efficacy relative to either agent alone in an LGSC PDX model with wildtype KRAS and with a RAF1 mutation. These data support an ongoing registration-directed study with avutometinib ± the FAK inhibitor defactinib for patients with recurrent LGSC (NCT04625270). Citation Format: Blair McNamara, Cem Demirkiran, Stefania Bellone, Levent Mutlu, Margherita Zipponi, Miguel S. Verzosa, Justin Harold, Tobias M. Hartwich, Gary Altwerger, Elena Ratner, Gloria Huang, Mitchell Clark, Vaagn Andikyan, Masoud Azodi, Peter E. Schwartz, Peter Dottino, Alessandro D. Santin. In vivo efficacy of RAF/MEK clamp avutometinib (VS-6766) in combination with FAK inhibition in low grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3402.

Abstract 2848: DNA replication stress and mitotic catastrophe mediate sotorasib addiction in KRASG12C-mutant cancer

Abstract The KRASG12C inhibitor sotorasib was approved by the US Food and Drug Administration (FDA) for treating KRASG12Cmutant locally advanced or metastatic NSCLC in 2021 (Nakajima, Drezner et al. 2022). Clinical trials on the use of sotorasib for cancer treatment have demonstrated promising results. However, KRASG12C-mutant cancers can acquire resistance to sotorasib after treatment. Most of the drug resistance mechanisms converge on reactivation of the MAPK pathway to bypass KRAS inhibition (Adachi, Ito et al. 2020, Ryan, Fece de la Cruz et al. 2020, Xue, Zhao et al. 2020, Zhao, Murciano-Goroff et al. 2021, Liu, Kang et al. 2022). Identifying the underlying drug resistance mechanisms is critical for improving treatment response and developing more effective combination strategies. During study on the mechanisms underlying sotorasib resistance, we incidentally found that when cultured in sotorasib-free medium, KRASG12C-mutant cancer cells adopted to sotorasib demonstrated a decreased growth rate and increased cell death. A similar “drug addiction” phenomenon was noted in BRAF-mutant cells treated with BRAF or MEK inhibitors (Das Thakur, Salangsang et al. 2013, Moriceau, Hugo et al. 2015, Hong, Moriceau et al. 2018, Rao, Shi et al. 2020). Here, we first verified the death property induced by sotorasib withdrawal in sotorasib-resistant cells, and further explored the mechanism underlying sotorasib addiction. Finally, we provide a novel therapeutic strategy against sotorasib-addicted cells. In the results, we uncovered in the absence of sotorasib, the sotorasib-resistant cells underwent p21Waf1/Cip1-mediated cell cycle arrest and caspase-dependent cell death. Robust mitogen-activated protein kinase (MAPK) pathway activation after sotorasib withdrawal triggered severe DNA damage and replication stress, resulting in DNA damage response (DDR) activation. Persistent MAPK pathway hyperactivation concomitant with exhausted DDR eventually drove the premature entry of cells into mitosis and to aberrant mitosis, followed by micronucleus and nucleoplasmic bridge formation. Pharmacologic activation of the MAPK pathway with a type I BRAF inhibitor could further enhance the effects of sotorasib withdrawal in sotorasib-resistant cancer cells in vitro and in vivo. Collectively, we identified the sotorasib addiction phenomenon in cancer cells, determined the underlying mechanisms of replication stress and mitotic catastrophe, and provide a novel therapeutic strategy against sotorasib-addicted cells with pharmacological enhancement of aberrant MAPK activation with a type I BRAF inhibitor, which can more effectively restrained cell growth both in vitro and in vivo. Citation Format: Li-Wen Chiou, Chien-Hui Chan, Yu-Ling Jhuang, Ching-Yao Yang, Yung-Ming Jeng. DNA replication stress and mitotic catastrophe mediate sotorasib addiction in KRASG12C-mutant cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2848.

Enhanced Apoptosis and Loss of Cell Viability in Melanoma Cells by Combined Inhibition of ERK and Mcl-1 Is Related to Loss of Mitochondrial Membrane Potential, Caspase Activation and Upregulation of Proapoptotic Bcl-2 Proteins.

Targeting of MAP kinase pathways by BRAF inhibitors has evolved as a key therapy for BRAF-mutated melanoma. However, it cannot be applied for BRAF-WT melanoma, and also, in BRAF-mutated melanoma, tumor relapse often follows after an initial phase of tumor regression. Inhibition of MAP kinase pathways downstream at ERK1/2, or inhibitors of antiapoptotic Bcl-2 proteins, such as Mcl-1, may serve as alternative strategies. As shown here, the BRAF inhibitor vemurafenib and the ERK inhibitor SCH772984 showed only limited efficacy in melanoma cell lines, when applied alone. However, in combination with the Mcl-1 inhibitor S63845, the effects of vemurafenib were strongly enhanced in BRAF-mutated cell lines, and the effects of SCH772984 were enhanced in both BRAF-mutated and BRAF-WT cells. This resulted in up to 90% loss of cell viability and cell proliferation, as well as in induction of apoptosis in up to 60% of cells. The combination of SCH772984/S63845 resulted in caspase activation, processing of poly (ADP-ribose) polymerase (PARP), phosphorylation of histone H2AX, loss of mitochondrial membrane potential, and cytochrome c release. Proving the critical role of caspases, a pan-caspase inhibitor suppressed apoptosis induction, as well as loss of cell viability. As concerning Bcl-2 family proteins, SCH772984 enhanced expression of the proapoptotic Bim and Puma, as well as decreased phosphorylation of Bad. The combination finally resulted in downregulation of antiapoptotic Bcl-2 and enhanced expression of the proapoptotic Noxa. In conclusion, combined inhibition of ERK and Mcl-1 revealed an impressive efficacy both in BRAF-mutated and WT melanoma cells, and may thus represent a new strategy for overcoming drug resistance.

Overexpression of the pro-protein convertase furin predicts prognosis and promotes papillary thyroid carcinoma progression and metastasis through RAF/MEK signaling.

Furin belongs to the pro-protein convertases (PCs) family and its aberrant expression has been documented in various types of cancers; however, its role in thyroid cancer remains unclear. We investigated the expression of furin in a large cohort of Middle Eastern papillary thyroid carcinoma (PTC) patient samples and explored its functional role and mechanism in PTC cell lines in vitro and in vivo. Furin overexpression was observed in 44.6% of all PTC cases and was significantly associated with aggressive clinicopathological parameters and poor outcomes. We show that the knockdown of FURIN suppresses tumor growth, proliferation, migration, invasion, spheroid growth, and progression of epithelial-to-mesenchymal transition (EMT) in B-Raf proto-oncogene, serine/threonine kinase (BRAF) mutant cells, whereas its overexpression in BRAF wild-type PTC cell lines reversed the effect. FURIN knockdown in the BRAF mutant cell line led to reduced tumor growth and increased apoptosis. Mechanistically, FURIN knockdown led to MEK/ERK pathway suppression in BRAF mutant cells, although inhibition of MEK did not affect furin expression, which suggests that furin acts through the MEK/ERK pathway. Furthermore, our study revealed the synergistic antitumor effect of furin depletion and anti-MEK inhibitor treatment. Overall, these results indicate that furin is an important prognostic marker in Middle Eastern PTC and that it plays a crucial role in BRAF-associated MAP/ERK pathway activation and tumorigenesis. Furin inhibition could be a potential therapeutic target for aggressive PTC.

Design, synthesis and characterisation of a novel type II B-RAF paradox breaker inhibitor

The mutation V600E in B-Raf leads to mitogen activated protein kinase (MAPK) pathway activation, uncontrolled cell proliferation, and tumorigenesis. ATP competitive type I B-Raf inhibitors, such as vemurafenib (1) and PLX4720 (4) efficiently block the MAPK pathways in B-Raf mutant cells, however these inhibitors induce conformational changes in the wild type B-Raf (wtB-Raf) kinase domain leading to heterodimerization with C-Raf, causing paradoxical hyperactivation of the MAPK pathway. This unwanted activation may be avoided by another class of inhibitors (type II) which bind the kinase in the DFG-out conformation, such as AZ628 (3) preventing heterodimerization. Here we present a new B-Raf kinase domain inhibitor, based on a phenyl(1H-pyrrolo [2,3-b]pyridin-3-yl)methanone template, that represents a hybrid between 4 and 3. This novel inhibitor borrows the hinge binding region from 4 and the back pocket binding moiety from 3. We determined its binding mode, performed activity/selectivity studies, and molecular dynamics simulations in order to study the conformational effects induced by this inhibitor on wt and V600E mutant B-Raf kinase. We discovered that the inhibitor was active and selective for B-Raf, binds in a DFG-out/αC-helix-in conformation, and did not induce the aforementioned paradoxical hyperactivation in the MAPK pathway. We propose that this merging approach can be used to design a novel class of B-Raf inhibitors for translational studies.

Disulfiram/Cu Kills and Sensitizes BRAF-Mutant Thyroid Cancer Cells to BRAF Kinase Inhibitor by ROS-Dependently Relieving Feedback Activation of MAPK/ERK and PI3K/AKT Pathways

BRAFV600E, the most common genetic alteration, has become a major therapeutic target in thyroid cancer. Vemurafenib (PLX4032), a specific inhibitor of BRAFV600E kinase, exhibits antitumor activity in patients with BRAFV600E-mutated thyroid cancer. However, the clinical benefit of PLX4032 is often limited by short-term response and acquired resistance via heterogeneous feedback mechanisms. Disulfiram (DSF), an alcohol-aversion drug, shows potent antitumor efficacy in a copper (Cu)-dependent way. However, its antitumor activity in thyroid cancer and its effect on cellular response to BRAF kinase inhibitors remain unclear. Antitumor effects of DSF/Cu on BRAFV600E-mutated thyroid cancer cells and its effect on the response of these cells to BRAF kinase inhibitor PLX4032 were systematically assessed by a series of in vitro and in vivo functional experiments. The molecular mechanism underlying the sensitizing effect of DSF/Cu on PLX4032 was explored by Western blot and flow cytometry assays. DSF/Cu exhibited stronger inhibitory effects on the proliferation and colony formation of BRAFV600E-mutated thyroid cancer cells than DSF treatment alone. Further studies revealed that DSF/Cu killed thyroid cancer cells by ROS-dependent suppression of MAPK/ERK and PI3K/AKT signaling pathways. Our data also showed that DSF/Cu strikingly increased the response of BRAFV600E-mutated thyroid cancer cells to PLX4032. Mechanistically, DSF/Cu sensitizes BRAF-mutant thyroid cancer cells to PLX4032 by inhibiting HER3 and AKT in an ROS-dependent way and subsequently relieving feedback activation of MAPK/ERK and PI3K/AKT pathways. This study not only implies potential clinical use of DSF/Cu in cancer therapy but also provides a new therapeutic strategy for BRAFV600E-mutated thyroid cancers.

Hyperforin Elicits Cytostatic/Cytotoxic Activity in Human Melanoma Cell Lines, Inhibiting Pro-Survival NF-κB, STAT3, AP1 Transcription Factors and the Expression of Functional Proteins Involved in Mitochondrial and Cytosolic Metabolism.

Hyperforin (HPF), the main component responsible for the antidepressant action of Hypericum perforatum, displays additional beneficial properties including anti-inflammatory, antimicrobic, and antitumor activities. Among its antitumor effects, HPF activity on melanoma is poorly documented. Melanoma, especially BRAF-mutated melanoma, is still a high-mortality tumor type and the currently available therapies do not provide solutions. We investigated HPF's antimelanoma effectiveness in A375, FO1 and SK-Mel-28 human BRAF-mutated cell lines. Cell viability assays documented that all melanoma cells were affected by low HPF concentrations (EC50% 2-4 µM) in a time-dependent manner. A Br-deoxy-uridine incorporation assay attested a significant reduction of cell proliferation accompanied by decreased expression of cyclin D1 and A2, CDK4 and of the Rb protein phosphorylation, as assessed by immunoblots. In addition, the expression of P21/waf1 and the activated form of P53 were increased in A375 and SK-Mel-28 cells. Furthermore, HPF exerts cytotoxic effects. Apoptosis is induced 24 h after HPF administration, documented by an increase of cleaved-PARP1 and a decrease of both Bcl2 and Bcl-xL expression levels. Autophagy is induced, attested by an augmented LC3B expression and augmentation of the activated form of AMPK. Moreover, HPF lowers GPX4 enzyme expression, suggesting ferroptosis induction. HPF has been reported to activate the TRPC6 Ca++ channel and/or Ca++ and Zn++ release from mitochondria stores, increasing cytosolic Ca++ and Zn++ concentrations. Our data highlighted that HPF affects many cell-signaling pathways, including signaling induced by Ca++, such as FRA1, pcJun and pCREB, the expression or activity of which are increased shortly after treatment. However, the blockage of the TRPC6 Ca++ channel or the use of Ca++ and Zn++ chelators do not hinder HPF cytostatic/cytotoxic activity, suggesting that damages induced in melanoma cells may pass through other pathways. Remarkably, 24 h after HPF treatment, the expression of activated forms of the transcription factors NF-κB P65 subunit and STAT3 are significantly lowered. Several cytosolic (PGM2, LDHA and pPKM2) and mitochondrial (UQCRC1, COX4 and ATP5B) enzymes are downregulated by HPF treatment, suggesting a generalized reduction of vital functions in melanoma cells. In line with these results is the recognized ability of HPF to affect mitochondrial membrane potential by acting as a protonophore. Finally, HPF can hinder both melanoma cell migration and colony formation in soft agar. In conclusion, we provide evidence of the pleiotropic antitumor effects induced by HPF in melanoma cells.

Yap governs a lineage-specific neuregulin1 pathway-driven adaptive resistance to RAF kinase inhibitors

BackgroundInactivation of the Hippo pathway promotes Yap nuclear translocation, enabling execution of a transcriptional program that induces tissue growth. Genetic lesions of Hippo intermediates only identify a minority of cancers with illegitimate YAP activation. Yap has been implicated in resistance to targeted therapies, but the mechanisms by which YAP may impact adaptive resistance to MAPK inhibitors are unknown.MethodsWe screened 52 thyroid cancer cell lines for illegitimate nuclear YAP localization by immunofluorescence and fractionation of cell lysates. We engineered a doxycycline (dox)-inducible thyroid-specific mouse model expressing constitutively nuclear YAPS127A, alone or in combination with endogenous expression of either HrasG12V or BrafV600E. We also generated cell lines expressing dox-inducible sh-miR-E-YAP and/or YAPS127A. We used cell viability, invasion assays, immunofluorescence, Western blotting, qRT-PCRs, flow cytometry and cell sorting, high-throughput bulk RNA sequencing and in vivo tumorigenesis to investigate YAP dependency and response of BRAF-mutant cells to vemurafenib.ResultsWe found that 27/52 thyroid cancer cell lines had constitutively aberrant YAP nuclear localization when cultured at high density (NU-YAP), which rendered them dependent on YAP for viability, invasiveness and sensitivity to the YAP-TEAD complex inhibitor verteporfin, whereas cells with confluency-driven nuclear exclusion of YAP (CYT-YAP) were not. Treatment of BRAF-mutant thyroid cancer cells with RAF kinase inhibitors resulted in YAP nuclear translocation and activation of its transcriptional output. Resistance to vemurafenib in BRAF-mutant thyroid cells was driven by YAP-dependent NRG1, HER2 and HER3 activation across all isogenic human and mouse thyroid cell lines tested, which was abrogated by silencing YAP and relieved by pan-HER kinase inhibitors. YAP activation induced analogous changes in BRAF melanoma, but not colorectal cells.ConclusionsYAP activation in thyroid cancer generates a dependency on this transcription factor. YAP governs adaptive resistance to RAF kinase inhibitors and induces a gene expression program in BRAFV600E-mutant cells encompassing effectors in the NRG1 signaling pathway, which play a central role in the insensitivity to MAPK inhibitors in a lineage-dependent manner. HIPPO pathway inactivation serves as a lineage-dependent rheostat controlling the magnitude of the adaptive relief of feedback responses to MAPK inhibitors in BRAF-V600E cancers.

Sensitivities and Dependencies of BRAF Mutant Colorectal Cancer Cell Lines with or without PIK3CA Mutations for Discovery of Vulnerabilities with Therapeutic Potential.

Background: Colorectal cancer represents a common malignancy and remains incurable in the metastatic stage. Identification of molecular alterations that are present in colorectal cancer has led to the introduction of targeted therapies that improve outcomes. BRAF and PIK3CA mutations are observed in a subset of colorectal cancers. Colorectal cancers bearing BRAF mutations may be treated with specific BRAF inhibitors. These drugs benefit patients with BRAF mutant colorectal cancers but responses are rather brief, and progression is the rule. In contrast, no PI3K inhibitors have proven successful yet in the disease. Thus, new treatments to supplement the currently available drugs would be welcome to further improve survival. Methods: Profiled colorectal cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) were examined for BRAF and PIK3CA mutations and were interrogated for molecular characteristics and concomitant alterations that mirror clinical sample alterations. The Genomics of Drug Sensitivity in Cancer (GDSC) project was used for determination of drug sensitivities of BRAF mutated colorectal cell lines with or without concomitant PIK3CA mutations. The Cancer Dependency Map project served as the basis for identification of molecular dependencies and vulnerabilities in these cell lines. Results: CCLE includes 84 colorectal cancer cell lines, which recapitulate the molecular landscape of colorectal cancer. Of these, 23 and 24 cell lines possess BRAF and PIK3CA mutations, respectively. Seven BRAF mutant cell lines have V600E mutations and 14 PIK3CA mutant cell lines have hotspot helical or kinase domain mutations. V600E BRAF mutant cell lines with or without hotspot PIK3CA mutations are heterogeneous in their MSI status and mimic colorectal cancer tissues in other prevalent abnormalities including APC and TP53 mutations. Essential genes for survival include CTNNB1, WRN, and pyrimidine metabolism enzyme CAD. Besides BRAF mutations, BRAF inhibitor sensitivity in colorectal cancer cell lines is conferred by SACS mutations and PRKN locus loss. Conclusions: Colorectal cancer cell lines bearing the frequent BRAF and PIK3CA mutations present many alterations of the parental cancer tissue. Described vulnerabilities represent leads for therapeutic exploration in colorectal cancers with the corresponding alterations.

Head-to-Head Comparison of BRAF/MEK Inhibitor Combinations Proposes Superiority of Encorafenib Plus Trametinib in Melanoma

Simple SummaryA decade ago, the diagnosis of metastatic melanoma was mostly a death sentence. This has changed since new therapies became widely available in the clinical setting. In addition to checkpoint inhibitors, targeted therapy with BRAF and MEK inhibitors is standard care for BRAF-mutated melanoma, which accounts for almost half of all melanoma cases. The second largest group of melanoma patients, whose tumors harbor a mutation in the NRAS gene, demonstrates only a limited response to targeted therapy with MEK inhibitors. The aim of this investigation was to directly compare all possible BRAF/MEK inhibitor combinations in addition to the currently applied regimens. The analyzed data suggested that the combination of the BRAF inhibitor encorafenib and the MEK inhibitor trametinib demonstrated the highest anti-tumor activity in both, BRAF- and NRAS-mutated melanoma. This combination is not presently used in patient treatment, and therefore, deserves an opportunity to become part of clinical trials.BRAFV600 mutations in melanoma are targeted with mutation-specific BRAF inhibitors in combination with MEK inhibitors, which have significantly increased overall survival, but eventually lead to resistance in most cases. Additionally, targeted therapy for patients with NRASmutant melanoma is difficult. Our own studies showed that BRAF inhibitors amplify the effects of MEK inhibitors in NRASmutant melanoma. This study aimed at identifying a BRAF and MEK inhibitor combination with superior anti-tumor activity to the three currently approved combinations. We, thus, assessed anti-proliferative and pro-apoptotic activities of all nine as well as resistance-delaying capabilities of the three approved inhibitor combinations in a head-to-head comparison in vitro. The unconventional combination encorafenib/trametinib displayed the highest activity to suppress proliferation and induce apoptosis, acting in an additive manner in BRAFmutant and in a synergistic manner in NRASmutant melanoma cells. Correlating with current clinical studies of approved inhibitor combinations, encorafenib/binimetinib prolonged the time to resistance most efficiently in BRAFmutant cells. Conversely, NRASmutant cells needed the longest time to establish resistance when treated with dabrafenib/trametinib. Together, our data indicate that the most effective combination might not be currently used in clinical settings and could lead to improved overall responses.