Targeting MEK Research Articles

Androgen receptor blockade promotes response to BRAF/MEK-targeted therapy.

Treatment with therapy targeting BRAF and MEK (BRAF/MEK) has revolutionized care in melanoma and other cancers; however, therapeutic resistance is common and innovative treatment strategies are needed1,2. Here we studied a group of patients with melanoma who were treated with neoadjuvant BRAF/MEK-targeted therapy ( NCT02231775 , n = 51) and observed significantly higher rates of major pathological response (MPR; ≤10% viable tumour at resection) and improved recurrence-free survival (RFS) in female versus male patients (MPR, 66% versus 14%, P = 0.001; RFS, 64% versus 32% at 2 years, P = 0.021). The findings were validated in several additional cohorts2-4 of patients with unresectable metastatic melanoma who were treated with BRAF- and/or MEK-targeted therapy (n = 664 patients in total), demonstrating improved progression-free survival and overall survival in female versus male patients in several of these studies. Studies in preclinical models demonstrated significantly impaired anti-tumour activity in male versus female mice after BRAF/MEK-targeted therapy (P = 0.006), with significantly higher expression of the androgen receptor in tumours of male and female BRAF/MEK-treated mice versus the control (P = 0.0006 and P = 0.0025). Pharmacological inhibition of androgen receptor signalling improved responses to BRAF/MEK-targeted therapy in male and female mice (P = 0.018 and P = 0.003), whereas induction of androgen receptor signalling (through testosterone administration) was associated with a significantly impaired response to BRAF/MEK-targeted therapy in male and female patients (P = 0.021 and P < 0.0001). Together, these results have important implications for therapy.

Characterization of undescribed melanoma inhibitors from Euphorbia mauritanica L. cultivated in Egypt targeting BRAFV600E and MEK 1 kinases via in-silico study and ADME prediction

Three undescribed diterpenes including two ent-abietanes, euphomauritanol A, and euphomauritanol B, and one jatrophane, euphomauritanophane A, in addition to eight previously described metabolites were isolated from the MeOH–CH2Cl2 (1:1) extract of the Euphorbia mauritanica. The chemical structures of isolates were established based on the spectroscopic means including FT-IR, HRMS, 1D and 2D NMR. The absolute stereochemistry of the undescribed diterpenes was deduced by experimental and calculated TDDFT-electronic circular dichroism (ECD). The anti-proliferative effects of the isolated diterpenes were evaluated against B16-BL6, Hep G2, and Caco-2. The euphomauritanol A, euphomauritanol B, and euphomauritanophane A significantly inhibited the growth of murine melanoma B16-BL6 cell lines with IC50 10.28, 20.22, and 38.81 μM, respectively with no responses against the other cells. These activities were rationalized by molecular docking of the active compounds in BRAFV600E and MEK1 active sites. Moreover, the in-silico pharmacokinetics predictions by Swiss ADME revealed that the active compounds possessed favorable oral bioavailability and drug-likeness properties.

How I treat refractory/relapsed hairy cell leukemia with BRAF inhibitors

AbstractHairy cell leukemia (HCL) responds very well to frontline chemotherapy with purine analogs (cladribine and pentostatine). However, approximately half of patients experience 1 or more relapses, which become progressively resistant to these myelotoxic and immunosuppressive agents. At progression, standard therapeutic options include a second course of purine analogs alone or in combination with rituximab and, upon second relapse, therapy with the anti-CD22 immunotoxin moxetumomab pasudotox. Furthermore, blockade of the mutant BRAF-V600E kinase (the pathogenetic hallmark of HCL) through orally available specific inhibitors (vemurafenib or dabrafenib) effaces the peculiar morphologic, phenotypic, and molecular identity of this disease and its typical antiapoptotic behavior and is emerging as an attractive chemotherapy-free strategy in various clinical scenarios. These include patients with, or at risk of, severe infections and, in a highly effective combination with rituximab, patients with relapsed or refractory HCL. Other treatments explored in clinical trials are BTK inhibition with ibrutinib and co-inhibition of BRAF (through dabrafenib or vemurafenib) and its downstream target MEK (through trametinib or cobimetinib). Here, we focus on our experience with BRAF inhibitors in clinical trials and as off-label use in routine practice by presenting 3 challenging clinical cases to illustrate their management in the context of all available treatment options.

MEK inhibition overcomes resistance to EphA2-targeted therapy in uterine cancer

BackgroundOur pilot clinical study of EphA2 inhibitor (dasatinib) plus paclitaxel and carboplatin showed interesting clinical activity in endometrial cancer with manageable toxicity. However, the underlying mechanisms of dasatinib resistance in uterine cancer are unknown. Here, we investigated potential mechanisms underlying resistance to EphA2 inhibitors in uterine cancer and examined the anti-tumor activity of EphA2 inhibitors alone and in combination with a MEK inhibitor. MethodsWe evaluated the antitumor activity of EphA2 inhibitors plus a MEK inhibitor using in vitro and in vivo orthotopic models of uterine cancer. ResultsEphA2 inhibitor induced MAPK in dasatinib-resistant uterine cancer cells (HEC-1A and Ishikawa) and BRAF/CRAF heterodimerization in HEC-1A cells. EphA2 inhibitor and trametinib significantly increased apoptosis in cancer cells resistant to EphA2 inhibitors compared with controls (p < 0.01). An in vivo study with the orthotopic HEC-1A model showed significantly greater antitumor response to combination treatment compared with dasatinib alone (p < 0.01). Combination treatment increased EphrinA1 and BIM along with decreased pMAPK, Jagged 1, and c-MYC expression in dasatinib-resistant cells. In addition, Spearman analysis using the TCGA data revealed that upregulation of EphA2 was significantly correlated with JAG1, MYC, NOTCH1, NOTCH3 and HES1 expression (p < 0.001, r = 0.25–0.43). Specifically, MAP3K15 and the NOTCH family genes were significantly related to poor clinical outcome in patients with uterine cancer. ConclusionsThese findings indicate that the MAPK pathway is activated in dasatinib-resistant uterine cancer cells and that EphrinA1-mediated MEK inhibition overcomes dasatinib resistance. Dual targeting of both EphA2 and MEK, combined with chemotherapy, should be considered for future clinical development.

Dual targeting of MEK and PI3K effectively controls the proliferation of human EGFR-TKI resistant non-small cell lung carcinoma cell lines with different genetic backgrounds

BackgroundMolecular targeted therapy for non-small cell lung carcinoma (NSCLC) is restricted due to resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). This study evaluated the effects of dual targeting of MEK and PI3K in human EGFR-TKI resistant NSCLC cell lines.MethodsEGFR-TKI resistant NSCLC cell lines H1975, H460, and A549, with different mutation and amplification status in EGFR, K-RAS, PIK3CA, and MET genes, were treated with a MEK162 (MEK inhibitor) and BKM120 (PI3K inhibitor) combination or a BIBW2992 (EGFR inhibitor) and ARQ197 (MET inhibitor) combination and assayed for cell proliferation, apoptosis, and cell cycle distribution.ResultsDual targeting of MEK and PI3K efficiently inhibited the cell proliferation, induced apoptosis and the G0/G1 cell cycle, and decreased the phosphorylation of ERK1/2, AKT, S6, and 4E-BP1. H460 cells with K-RAS and PIK3CA mutation were most sensitive to MEK162 and BKM120 combinations. H1975 cells with EGFR and PIK3CA mutation and MET amplification were sensitive to BIBW2992 and ARQ197 combinations.ConclusionDual targeting regulated the proliferation of EGFR-TKI-resistant NSCLC cells, especially mutants in K-RAS and PIK3CA that are promising for EGFR-TKI-resistant NSCLC therapeutics.

Targeting Cell Non-Autonomous MAPK Activation As a Novel Therapeutic Strategy in Myeloproliferative Neoplasms

Activated JAK2 signaling is a central feature of myeloproliferative neoplasm (MPN) pathogenesis, and drives myeloid blood cell production through constitutive activation of STAT transcription factors, the PI3K/AKT and the MAPK signaling pathways. JAK2 inhibitors show significant, but limited clinical efficacy such as seen for ruxolitinib, a type I JAK1/2 inhibitor approved for the treatment of myelofibrosis (MF) and polycythemia vera (PV). Given the impact of JAK2 inhibition on the MAPK signaling pathway in MPN has not been studied in vivo, we hypothesized that MAPK signaling could contribute to MPN cell survival in the setting of JAK kinase inhibition.We observed that JAK2 inhibition by ruxolitinib and other agents suppressed phosphorylation of ERK1/2, the distal kinases in the MAPK pathway, in MPN cell lines. By contrast, these JAK2 inhibitors could not suppress MAPK signaling in vivo in Jak2 V617F- and MPL W515L-driven murine MPN models. In the in vivo setting, ERK remained fully activated despite JAK2 inhibition at therapeutic or higher doses suggesting that compensatory signaling may bypass JAK2 inhibition. E x vivo exposure of Jak2 V617F and MPL W515L primary mouse hematopoietic cells or PBMCs from JAK2 V617F mutant MPN patients to ruxolitinib demonstrated MAPK inhibition, indicating that cell extrinsic stimuli maintain ERK activation in vivo .To identify secreted factors which could mediate JAK-independent ERK activation in vivo, we performed receptor tyrosine kinase arrays in Jak2 V617F and MPL W515L mouse splenocytes in presence and absence of ruxolitinib. This allowed us to show that expression and activation of PDGFRα was maintained in MPN cells upon JAK inhibitor treatment. Although the production of multiple cytokines was reduced by ruxolitinib, PDGFA and PDGFB production were not attenuated by JAK2 inhibitor treatment. We confirmed that PDGF-BB was present at higher levels than PDGF-AA in bone marrow supernatant, spleen supernatant and serum suggesting it is the primary ligand activating MAPK signaling through PDGFRα. Stimulation of primary Jak2 V617F bone marrow and spleen cells with PDGF-BB ex vivo induced ERK activation in presence of ruxolitinib suggesting PDGF-BB can mediate bypass ERK activation.We next assessed the efficacy of combined JAK/MEK inhibition in vivo . Binimetinib, a clinical-grade MEK1/2 inhibitor, inhibited ERK phosphorylation in Jak2 V617F and MPL W515L murine models at a single dose or with extended treatment, and showed modest efficacy when given as monotherapy in vivo. Of note, dual targeting of JAK2 and MEK with combined ruxolitinib/binimetinib was superior to both single agent therapies. In the Jak2 V617F model of PV, dual JAK/MEK inhibition corrected splenomegaly and reduced the increased hematocrit, reticulocytes, erythroid and megakaryocytic-erythroid progenitor populations characteristic of this MPN model. Bone marrow and spleen fibrosis was resolved by dual JAK/MEK inhibition along with reduced bone marrow hypercellularity and decreased mutant allele burden. Expression of ERK downstream targets including ETV5, EGR1, CCND1, DUSP4, DUSP6 and SPRED2 was effectively reduced by combined JAK/MEK inhibitor therapy. Dual JAK/MEK inhibition with ruxolitinib/binimetinib also showed increased efficacy in the MPL W515L model of ET/MF with improved correction of leuko- and thrombocytosis, extramedullary hematopoiesis, hepato- and splenomegaly and megakaryocytic hyperplasia. Combined JAK/MEK inhibition with ruxolitinib/selumetinib showed similar effects. Importantly, dual JAK/MEK inhibition reversed fibrosis to an extent not seen with JAK inhibitor monotherapy.Taken together, these data demonstrate that JAK2 inhibition incompletely suppresses ERK activation in MPN in vivo and that cell non-autonomous ERK activation through PDGFRα contributes to the limited efficacy of JAK inhibition in MPN. We show that dual targeting of JAK2 and MEK provides improved therapeutic efficacy by preventing bypass ERK activation and suggest that dual JAK/MEK inhibition should be pursued as a therapeutic approach for MPN patients. Our data also suggest that cell non-autonomous MAPK activation might represent a mechanism of resistance to targeted therapies which is only observed in the in vivo setting and which can guide combination therapy trials in different malignant contexts. [Display omitted] DisclosuresRadimerski:Novartis: Employment. Skoda:Novartis: Consultancy, Speakers Bureau; Shire: Speakers Bureau; Baxalta: Consultancy, Speakers Bureau. Levine:Roche: Research Funding; Celgene: Research Funding; Celgene: Research Funding; Roche: Research Funding; Qiagen: Equity Ownership; Qiagen: Equity Ownership.

Combined Blockade of MEK and CDK4/6 Pathways Induces Senescence to Improve Survival in Pancreatic Ductal Adenocarcinoma.

Activating KRAS mutations, a defining feature of pancreatic ductal adenocarcinoma (PDAC), promote tumor growth in part through the activation of cyclin-dependent kinases (CDK) that induce cell-cycle progression. p16INK4a (p16), encoded by the gene CDKN2A, is a potent inhibitor of CDK4/6 and serves as a critical checkpoint of cell proliferation. Mutations in and subsequent loss of the p16 gene occur in PDAC at a rate higher than that reported in any other tumor type and results in Rb inactivation and unrestricted cellular growth. Therefore, strategies targeting downstream RAS pathway effectors combined with CDK4/6 inhibition (CDK4/6i) may have the potential to improve outcomes in this disease. Herein, we show that expression of p16 is markedly reduced in PDAC tumors compared with normal pancreatic or pre-neoplastic tissues. Combined MEK inhibition (MEKi) and CDK4/6i results in sustained downregulation of both ERK and Rb phosphorylation and a significant reduction in cell proliferation compared with monotherapy in human PDAC cells. MEKi with CDK4/6i reduces tumor cell proliferation by promoting senescence-mediated growth arrest, independent of apoptosis in vitro We show that combined MEKi and CDK4/6i treatment attenuates tumor growth in xenograft models of PDAC and improves overall survival over 200% compared with treatment with vehicle or individual agents alone in Ptf1acre/+ ;LSL-KRASG12D/+ ;Tgfbr2flox/flox (PKT) mice. Histologic analysis of PKT tumor lysates reveal a significant decrease in markers of cell proliferation and an increase in senescence-associated markers without any significant change in apoptosis. These results demonstrate that combined targeting of both MEK and CDK4/6 represents a novel therapeutic strategy to synergistically reduce tumor growth through induction of cellular senescence in PDAC.

The Potential Efficacy of β-Blockers on Melanoma Survival: A Narrative Review.

Melanoma is a common tumor accounting for around 3&ndash;5% of all cutaneous malignancies with worldwide increasing incidence. It is still associated with significant mortality despite the breakthrough of new innovative therapies within the last decade. A wide variety of treatment modalities is currently used for the management of melanoma, ranging from surgical excision of primary melanoma to adju-vant and palliative treatment with target molecules, including BRAF and MEK inhibitors, and immune checkpoint inhibitors. &beta;-blockers have recently demonstrated in preclinical and clinical studies to reduce recurrence and to correlate with better overall survival in meta-static melanoma as an additional supportive treatment option, owing to their anti-tumor potential. Further investigation regarding their efficacy and safety profile is needed, since there are only few studies in the literature on this topic. Our aim is to evaluate the role and current status of &beta;-blockers in melanoma management. The literature research includes peer-reviewed articles (clinical trials or scien-tific reviews). Studies were identified by searching electronic databases (MEDLINE and PubMed) till May 2020 and reference lists of respective articles. Only articles published in English language were included. J Drugs Dermatol. 20(4):380-383. doi:10.36849/JDD.5673.

Research progress of MEK1/2 inhibitors and degraders in the treatment of cancer.

Mitogen-activated protein kinase kinases 1 and 2 (MEK1/2) are the crucial part of the RAS-RAF-MEK-ERK pathway (or ERK pathway), which is involved in the regulation of various cellular processes including proliferation, survival, and differentiation etal. Targeting MEK has become an important strategy for cancer therapy, and 4 MEK inhibitors (MEKis) have been approved by FDA to date. However, the application of MEKis is limited due to acquired resistance under long-term treatment. Fortunately, an emerging technology, named proteolysis targeting chimera (PROTAC), could break through this limitation by inducing MEK1/2 degradation. Compared to MEKis, MEK1/2 PROTAC is rarely studied and only three MEK1/2 PROTAC molecules, have been reported until now. This paper will outline the ERK pathway and the mechanism and research progress of MEK1/2 inhibitors, but focus on the development of MEK degraders and their optimization strategies. PAC-1 strategy which can induce MEK degradation indirectly, other PROTACs on ERK pathway, the advantages and challenges of PROTAC technology will be subsequently discussed.

Drug Repurposing Screen on Patient-Derived Organoids Identifies New Therapeutic Drug Combination Against KRAS Mutant Colon Cancer

Patient-derived organoids (PDO) are widely heralded as a drug-screening platform to develop new anti-cancer therapies. Here we use a drug-repurposing library to screen PDOs of colorectal cancer (CRC) to identify drugs that enhance the efficacy of combinatorial targeting of EGFR and MEK in KRAS mutant CRCs. Although this combination achieves significant inhibition of downstream signaling of mutant KRAS, preclinical models revealed its failure to induce robust cell killing. We developed a microscopy-based screen to score drug-induced cytotoxicity in PDOs and tested 414 putative anti-cancer drugs in combination with EGFR/MEK inhibition. A majority of validated hits were microtubule targeting agents that are commonly used in clinical oncology, like taxanes and vinca-alkaloids. One of these drugs, vinorelbine, was consistently effective across a panel of >20 different CRC PDOs. Unlike vinorelbine alone, its combination with EGFR/MEK inhibition induces apoptosis at all stages of the cell cycle, presumably by disrupting the balance between pro- and anti-apoptotic effectors. In mice, the triple combination showed tolerability and superior antitumor activity at clinically relevant doses and scheduling, setting the basis for a clinical trial to treat patients with metastatic RAS mutant CRC.

The Potential Efficacy of β-Blockers on Melanoma Survival: A Narrative Review

Melanoma is a common tumor accounting for around 3–5% of all cutaneous malignancies with worldwide increasing incidence. It is still associated with significant mortality despite the breakthrough of new innovative therapies within the last decade. A wide variety of treatment modalities is currently used for the management of melanoma, ranging from surgical excision of primary melanoma to adju-vant and palliative treatment with target molecules, including BRAF and MEK inhibitors, and immune checkpoint inhibitors. β-blockers have recently demonstrated in preclinical and clinical studies to reduce recurrence and to correlate with better overall survival in meta-static melanoma as an additional supportive treatment option, owing to their anti-tumor potential. Further investigation regarding their efficacy and safety profile is needed, since there are only few studies in the literature on this topic. Our aim is to evaluate the role and current status of β-blockers in melanoma management. The literature research includes peer-reviewed articles (clinical trials or scien-tific reviews). Studies were identified by searching electronic databases (MEDLINE and PubMed) till May 2020 and reference lists of respective articles. Only articles published in English language were included. J Drugs Dermatol. 20(4):380-383. doi:10.36849/JDD.5673

Concomitant MEK and Cyclin Gene Alterations: Implications for Response to Targeted Therapeutics.

Cyclin and MAPK/MEK-related gene alterations are implicated in cell-cycle progression and cancer growth. Yet, monotherapy to target the cyclin (CDK4/6) or the MEK pathway has often yielded disappointing results. Because coalterations in cyclin and MEK pathway genes frequently cooccur, we hypothesized that resistance to CDK4/6 or MEK inhibitor monotherapy might be mediated via activation of oncogenic codrivers, and that combination therapy might be useful. Herein, we describe 9 patients with advanced malignancies harboring concomitant CDKN2A and/or CDKN2B alterations (upregulate CDK4/6) along with KRAS or BRAF alterations (activate the MEK pathway) who were treated with palbociclib (CDK4/6 inhibitor) and trametinib (MEK inhibitor) combination-based regimens. Two patients (with pancreatic cancer) achieved a partial remission (PR) and, overall, 5 patients (56%) had clinical benefit (stable disease ≥ 6 months/PR) with progression-free survival of approximately 7, 9, 9, 11, and 17.5+ months. Interestingly, 1 of these patients whose cancer (gastrointestinal stromal tumor) had progressed on MEK targeting regimen, did well for about 1 year after palbociclib was added. These observations suggest that cotargeting cyclin and MEK signaling can be successful when tumors bear genomic coalterations that activate both of these pathways. Further prospective studies using this matching precision strategy to overcome resistance are warranted.See related commentary by Groisberg and Subbiah, p. 2672.

New perspectives on targeting RAF, MEK and ERK in melanoma.

Although immune checkpoint inhibitors and small molecule inhibitors targeting the MAPK pathway have revolutionized the management of metastatic melanoma, long-term disease control occurs only for a minority of patients because of multiple resistance mechanisms. One way to tackle resistance is to develop the next-generation of RAF, MEK and ERK inhibitors using our understanding of the molecular mechanisms that fine-tune the MAPK pathway. Studies on the regulation of the MAPK pathway have revealed a dominant role for homo-dimerization and hetero-dimerization of RAF, MEK and ERK. Allosteric inhibitors that break these dimers are, therefore, undergoing various stages of preclinical and clinical evaluation. Novel MEK inhibitors are less susceptible to differences in MEK's activation state and do not drive the compensatory activation of MEK that could limit efficacy. Innovations in targeting ERK originate from dual inhibitors that block MEK-catalyzed ERK phosphorylation, thereby limiting the extent of ERK reactivation following feedback relief. The primary goal in RAF, MEK and ERK inhibitors' development is to produce molecules with less inhibitor paradox and off-target effects, giving robust and sustained MAPK pathway inhibition.

Abstract PO-035: Overcoming adaptive resistance to mutant KRAS and MEK inhibitors by co-targeting mTORC1/2 in pancreatic cancer

Abstract Activating KRAS mutations are found in over 90% of pancreatic ductal adenocarcinomas (PDAC), yet KRAS has remained a difficult target to inhibit pharmacologically. While mutant KRAS-specific inhibitors have recently been developed, there is increasing awareness that adaptive resistance to these inhibitors limits their efficacy as single agents. Here, we demonstrate, using several human and mouse models of PDAC, rapid acquisition of tumor resistance in response to genetic or pharmacologic targeting of KRAS or MEK, associated with increased phosphorylation of Akt and Rictor, in an ILK-dependent manner. While pharmacologic inhibition ofmTORC1/2 results in a compensatory increase in ERK phosphorylation, combinatorial treatment of PDAC cells with either KRAS or MEK inhibitors together with mTORC1/2 inhibitors results in synergistic cytotoxicity and cell death reflected by sustained inhibition of p-ERK and p-Rictor/p-Akt, and of downstream regulators of protein synthesis and cell survival. Importantly, relative to single agents alone, this combination leads to significant inhibition of tumor growth in vivo, including durable tumor regressions and increased survival. We have identified a highly effective combinatorial treatment strategy, using clinically viable inhibitors, which can be applied to PDAC tumors with different KRAS mutations. Citation Format: Wells Brown, Paul McDonald, Shoukat Dedhar. Overcoming adaptive resistance to mutant KRAS and MEK inhibitors by co-targeting mTORC1/2 in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-035.

Blocking Y-Box Binding Protein-1 through Simultaneous Targeting of PI3K and MAPK in Triple Negative Breast Cancers.

Simple SummaryTriple-negative breast cancer (TNBC) is associated with the high rates of relapse and metastasis and poor survival. YB-1 is overexpressed in TNBC tumor tissues. In the present study, we demonstrated that S102 phosphorylation of YB-1 in TNBC cell lines depend on the mutation status of the components of the MAPK/ERK and PI3K/Akt pathways. Simultaneous targeting of MEK and PI3K was found to be the most effective approach to block YB-1 phosphorylation and to inhibit YB-1 dependent cell proliferation. YBX1 knockout was sufficient to block TNBC tumor growth.The multifunctional protein Y-box binding protein-1 (YB-1) regulates all the so far described cancer hallmarks including cell proliferation and survival. The MAPK/ERK and PI3K/Akt pathways are also the major pathways involved in cell growth, proliferation, and survival, and are the frequently hyperactivated pathways in human cancers. A gain of function mutation in KRAS mainly leads to the constitutive activation of the MAPK pathway, while the activation of the PI3K/Akt pathway occurs either through the loss of PTEN or a gain of function mutation of the catalytic subunit alpha of PI3K (PIK3CA). In this study, we investigated the underlying signaling pathway involved in YB-1 phosphorylation at serine 102 (S102) in KRAS(G13D)-mutated triple-negative breast cancer (TNBC) MDA-MB-231 cells versus PIK3CA(H1047R)/PTEN(E307K) mutated TNBC MDA-MB-453 cells. Our data demonstrate that S102 phosphorylation of YB-1 in KRAS-mutated cells is mainly dependent on the MAPK/ERK pathway, while in PIK3CA/PTEN-mutated cells, YB-1 S102 phosphorylation is entirely dependent on the PI3K/Akt pathway. Independent of the individual dominant pathway regulating YB-1 phosphorylation, dual targeting of MEK and PI3K efficiently inhibited YB-1 phosphorylation and blocked cell proliferation. This represents functional crosstalk between the two pathways. Our data obtained from the experiments, applying pharmacological inhibitors and genetic approaches, shows that YB-1 is a key player in cell proliferation, clonogenic activity, and tumor growth of TNBC cells through the MAPK and PI3K pathways. Therefore, dual inhibition of these two pathways or single targeting of YB-1 may be an effective strategy to treat TNBC.

Abstract 550: Determining efficacy of drug combinations identified by unbiased high throughput screening for RAS-mutated colorectal cancer

Abstract Background: Metastatic colorectal cancer (mCRC) is the second leading cause of cancer death in the US. The response rate to current systemic therapies is ~50% and most patients die within 2.5 years from diagnosis of metastasis. With ~5% of patients with mCRC benefiting from immunotherapies, effective therapies for ~95% of patients with mCRC are urgently required. Greater than 50% of patients with mCRC harbor mutations in the oncogenic driver RAS (KRAS, NRAS). As targeting RAS directly is technically challenging for the majority of RAS mutated sites, efforts have been concentrated on targeting MEK, a downstream mediator of RAS. However, targeting MEK with single agent therapy is ineffective in patients with mCRC. Aims: We aimed to perform unbiased high throughput screening (HTS) to identify drugs that, when combined to MEK inhibitors, would enhance their efficacy. Methods: We performed HTS with CRC cells grown in 2D and 3D cell culture using the MEK inhibitor trametinib as a backbone, and two different compound libraries composed of drugs either approved by the FDA or in clinical trials; 1) the NCI oncology set V, and, 2) a custom clinical compound set. Using the Bliss model of synergy, multiple compounds were identified to be synergistic with trametinib. In vitro and in vivo validation of synergy for a specific drug combination, dasatinib with trametinib, were performed. Results: Our unbiased HTS studies demonstrated that combining the SRC inhibitor dasatinib with trametinib was synergistic in both, 2D and 3D, CRC cell culture. This combination was further validated by MTT and colony formation assays. RPPA and western blot analyses of markers for apoptosis and various cell proliferation markers demonstrated that targeting SRC with MEK inhibits cell proliferation and increases cell death in multiple CRC cell lines as compared to single agents. In vivo studies in mice using RAS mutated CRC PDXs and cell lines with these drugs at clinically relevant doses, however, failed to demonstrate significant tumor growth inhibition or tumor regression. Conclusions: Although our unbiased HTS and other in vitro validation studies demonstrated that combining dasatinib and trametinib can enhance the efficacy of MEK targeted therapy in RAS mutated mCRC cells, careful in vivo preclinical studies failed to demonstrate enhanced efficacy of combining these two drugs. These studies highlight the need for performing careful in vivo preclinical studies as a prerequisite to translate in vitro findings into clinical studies. Further, our studies emphasize the importance for performing in vivo studies using drug doses that reflect clinically achievable doses in humans to identify therapies, either single or in combination, that may finally benefit our patients. This study also exemplifies the importance of publishing negative data so that others can pursue alternative strategies. Citation Format: Fan Fan, Reid Powell, Jason Roszik, Rui Wang, Xiangcang Ye, Clifford Stephan, Lee Ellis, Rajat Bhattacharya. Determining efficacy of drug combinations identified by unbiased high throughput screening for RAS-mutated colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 550.

Melanoma: Rational Design of Polyglutamic Acid Delivering an Optimized Combination of Drugs Targeting Mutated BRAF and MEK in Melanoma (Adv. Therap. 8/2020)

In article number 2000028 Ronit Satchi-Fainaro and co-workers present a novel polymeric system that simultaneously delivers a combination of drugs that target BRAF/ MEK mutations in melanoma. This system possesses an improved solubility, stability, and selective release by cathepsins in the tumor site. Hence, it exhibits synergistic activity, with superior efficacy and safety compared to each monodrug polymer conjugate and combinations of the free drugs.

MAPK Pathway Alterations Correlate with Poor Survival and Drive Resistance to Therapy in Patients with Lung Cancers Driven by ROS1 Fusions.

ROS1 tyrosine kinase inhibitors (TKI) provide significant benefit in lung adenocarcinoma patients with ROS1 fusions. However, as observed with all targeted therapies, resistance arises. Detecting mechanisms of acquired resistance (AR) is crucial to finding novel therapies and improve patient outcomes. ROS1 fusions were expressed in HBEC and NIH-3T3 cells either by cDNA overexpression (CD74/ROS1, SLC34A2/ROS1) or CRISPR-Cas9-mediated genomic engineering (EZR/ROS1). We reviewed targeted large-panel sequencing data (using the MSK-IMPACT assay) patients treated with ROS1 TKIs, and genetic alterations hypothesized to confer AR were modeled in these cell lines. Eight of the 75 patients with a ROS1 fusion had a concurrent MAPK pathway alteration and this correlated with shorter overall survival. In addition, the induction of ROS1 fusions stimulated activation of MEK/ERK signaling with minimal effects on AKT signaling, suggesting the importance of the MAPK pathway in driving ROS1 fusion-positive cancers. Of 8 patients, 2 patients harbored novel in-frame deletions in MEK1 (MEK1delE41_L54) and MEKK1 (MEKK1delH907_C916) that were acquired after ROS1 TKIs, and 2 patients harbored NF1 loss-of-function mutations. Expression of MEK1del or MEKK1del, and knockdown of NF1 in ROS1 fusion-positive cells activated MEK/ERK signaling and conferred resistance to ROS1 TKIs. Combined targeting of ROS1 and MEK inhibited growth of cells expressing both ROS1 fusion and MEK1del. We demonstrate that downstream activation of the MAPK pathway can mediate of innate acquired resistance to ROS1 TKIs and that patients harboring ROS1 fusion and concurrent downstream MAPK pathway alterations have worse survival. Our findings suggest a treatment strategy to target both aberrations.

Inactivation of NF1 Promotes Resistance to EGFR Inhibition in KRAS/NRAS/BRAFV600 -Wild-Type Colorectal Cancer.

Through the use of an unbiased, genome-scale CRISPR modifier screen, we identified NF1 suppression as a mechanism of resistance to EGFR inhibition in NRAS/KRAS/BRAFV600 -wild-type colorectal cancer cells. Reduced NF1 expression permitted sustained signaling through the MAPK pathway to promote cell proliferation in the presence of EGFR inhibition. Targeting of MEK in combination with EGFR inhibition leads to synergistic antiproliferative activity. Human KRAS/NRAS/BRAFV600 -wild-type colorectal cancer cell lines with NF1 mutations displayed reduced NF1 mRNA or protein expression and were resistant to EGFR blockade by gefitinib or cetuximab. Cooccurring loss-of-function mutations in PTEN were associated with resistance to dual EGFR/MEK inhibition but cotreatment with a PI3K inhibitor further suppressed proliferation. Loss of NF1 may be a useful biomarker to identify patients that are less likely to benefit from single-agent anti-EGFR therapy in colorectal cancer and may direct potential combination strategies. IMPLICATIONS: This study suggests that further clinical validation of NF1 status as predictor of response to anti-EGFR targeting antibodies in patients with colorectal cancer with KRAS/NRAS/BRAFV600 -wild-type tumors is warranted.

Rational Design of Polyglutamic Acid Delivering an Optimized Combination of Drugs Targeting Mutated BRAF and MEK in Melanoma.

Targeted therapies against cancer can relieve symptoms and induce remission, however, they often present limited duration of disease control, cause side effects and often induce acquired resistance. Therefore, there is a great motivation to develop a unique delivery system, targeted to the tumor, in which we can combine several active entities, increase the therapeutic index by reducing systemic exposure, and enhance their synergistic activity. To meet these goals, we chose the biocompatible and biodegradable poly(α,L-glutamic acid) (PGA) as a nanocarrier that facilitates extravasation-dependent tumor targeting delivery. The RAS/RAF/MEK/ERK pathway when aberrantly activated in melanoma, can lead to uncontrolled cell proliferation, induced invasion, and reduced apoptosis. Here, we selected two drugs targeting this pathway; a MEK1/2 inhibitor (selumetinib; SLM) and a modified BRAF inhibitor (modified dabrafenib; mDBF), that exhibited synergism in vitro. We synthesized and characterized our nanomedicine of PGA conjugated to SLM and mDBF (PGA-SLM-mDBF). PGA-SLM-mDBF inhibited the proliferation of melanoma cells and decreased their migratory and sprouting abilities without inducing a hemolytic effect. Moreover, the polymer-2-drugs conjugate exhibited superior anti-tumor activity in comparison with the two separate polymer-drug conjugates in vitro and with free drugs in a mouse model of primary melanoma and prolonged survival at a lower dose.