Hyperactivation Of ERK Research Articles

Abstract PS12-07: Phase II clinical trial of palbociclib and binimetinib in advanced triple-negative breast cancer (TNBC) with hyperactivation of ERK and/or CDK4/6

Abstract Background: We previously demonstrated that a kinase-based taxonomy of TNBC was most parsimonious than next-generation sequencing in defining TNBC subtypes associated with prognostic categories in early disease. The most aggressive TNBC variants were driven by a heterogeneous set of genetic aberrations that converged in the increased activity of 6 kinases: KIT, PNKP, PRKCE, P70S6K, ERK and CDK6 (Nat Commun; 9:3501-18). The combined inhibition of these kinases in pairs led to potent tumor regression in preclinical models, being the most powerful combination that one directed against CDK6 and ERK. This prompted us to design a phase II trial testing the combination of palbociclib (against CDK6) and binimetinib (against the ERK upstream kinase MEK, since no ERK inhibitor was available at that moment outside phase I trials) in advanced TNBC. Hyperactivation of CDK6 and/or ERK was selected as entry criterion. Trial design: This was a single-arm, prospective, multicentric, open-label, phase II investigator-initiated trial. CDK6 and phosphor-ERK levels were measured in tumor samples by immunohistochemistry and normalized with a reference sample collection to a Z-score. Patients with scores for either kinase above the median were candidates for the trial. Key inclusion criteria included metastatic >18year-old TNBC, adequate organ function, measurable disease, and progression to 1-2 prior treatment lines (including immunotherapy, and a PARP inhibitor in case of germline BRCA1/2 mutation). Patients started continuous oral binimetinib at 45 mg/BID and palbociclib 100 mg/day from days 1 to 21, in 28-day cycles. Patients experiencing ≤ grade 1 tolerable side effects as the greatest toxicity were escalated to palbociclib to 125 in cycle 2. RECIST 1.1 and NCI CTC AE V 5.0 were used for assessing disease control (q8 weeks) and toxicity. The primary aims were to assess the efficacy and toxicity of this combination, and the secondary one to detect biomarkers of activity. At the time of trial design, in absence of available Sacituzumab for prescription, the reference PFS to beat in advanced lines for physician’s choice in TNBC was 1.7 month (NEJM; 384:1529-41, 2021). With alpha and beta errors of 0.05 and 0.2, the minimum number of patients to demonstrate a 30% improvement in PFS to 2.5 months was 25. Results: From November 2020 to April 2023, 69 patients were screened and 24 entered the trial (5 positive for phosphor-ERK; 2 for CDK6; 17 for both). Toxicity was generally mild and included grade 1-2 diarrhea (33% of the patients), grade 1-2 asthenia (50%), grade 1-3 neutropenia (75%), grade 2 retinal toxicity (8.3%) and grade 3 rash (4.2%); no grade 4/5 toxicities were observed. Median PFS was 1.83 months (range 0.3 to 11.3+). Phospho-ERK and CDK6 levels were not correlated (Pearson’s R= -0.089; P=0.68); CDK6 levels did not show association with PFS time (R = -0.120; P=0.58). Interestingly, however, phosphor-ERK levels in the baseline tumor sample showed correlation with PFS time (R = 0.428; P=0.037). Conclusion: The combination of palbociclib and binimetinib was generally safe, and PFS time showed correlation with baseline phosphorylation levels of ERK. However, the trial did not meet its primary endpoint. Citation Format: Rodrigo Sánchez-Bayona, Alfonso Cortes, Juan Miguel Cejalvo, Luis Manso, Serafin Morales, Jose A Garcia-Saenz, Jorge Silva, Juan A Guerra, Diego Malón-Giménez, Silvana Mouron, Eduardo Caleiras, Miguel Quintela-Fandino. Phase II clinical trial of palbociclib and binimetinib in advanced triple-negative breast cancer (TNBC) with hyperactivation of ERK and/or CDK4/6 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS12-07.

E-cadherin interacts with EGFR resulting in hyper-activation of ERK in multiple models of breast cancer.

The loss of intercellular adhesion molecule E-cadherin is a hallmark of the epithelial-mesenchymal transition (EMT), during which tumor cells transition into an invasive phenotype. Accordingly, E-cadherin has long been considered a tumor suppressor gene; however, E-cadherin expression is paradoxically correlated with breast cancer survival rates. Using novel multi-compartment organoids and multiple in vivo models, we show that E-cadherin promotes a hyper-proliferative phenotype in breast cancer cells via interaction with the transmembrane receptor EGFR. The E-cad and EGFR interaction results in activation of the MEK/ERK signaling pathway, leading to a significant increase in proliferation via activation of transcription factors, including c-Fos. Pharmacological inhibition of MEK activity in E-cadherin positive breast cancer significantly decreases both tumor growth and macro-metastasis in vivo. This work provides evidence for a novel role of E-cadherin in breast tumor progression and identifies a new target to treat hyper-proliferative E-cadherin-positive breast tumors, thus providing the foundation to utilize E-cadherin as a biomarker for specific therapeutic success.

ERK hyperactivation serves as a unified mechanism of escape in intrinsic and acquired CDK4/6 inhibitor resistance in acral lentiginous melanoma.

Patients with metastatic acral lentiginous melanoma (ALM) suffer worse outcomes relative to patients with other forms of cutaneous melanoma (CM), and do not benefit as well to approved melanoma therapies. Identification of cyclin-dependent kinase 4 and 6 (CDK4/6) pathway gene alterations in >60% of ALMs has led to clinical trials of the CDK4/6 inhibitor (CDK4i/6i) palbociclib for ALM; however, median progression free survival with CDK4i/6i treatment was only 2.2 months, suggesting existence of resistance mechanisms. Therapy resistance in ALM remains poorly understood; here we report hyperactivation of MAPK signaling and elevated cyclin D1 expression serve as a mechanism of intrinsic early/adaptive CDK4i/6i resistance. ALM cells that have acquired CDK4i/6i resistance following chronic treatment exposure also exhibit hyperactivation of the MAPK pathway. MEK and/or ERK inhibition increases CDK4i/6i efficacy against therapy naïve and CDK4i/6i-resistant AM cells in xenograft and patient-derived xenograft (PDX) models and promotes a defective DNA repair, cell cycle arrested and apoptotic program. Notably, gene alterations poorly correlate with protein expression of cell cycle proteins in ALM or efficacy of CDK4i/6i, urging additional strategies when stratifying patients for CDK4i/6i trial inclusion. Concurrent targeting of the MAPK pathway and CDK4/6 represents a new approach for patients with metastatic ALM to improve outcomes.

Exploring the In Vitro and In Vivo Therapeutic Potential of BRAF and MEK Inhibitor Combination in NRAS-Mutated Melanoma.

Patients with NRAS-mutant metastatic melanoma often have an aggressive disease requiring a fast-acting, effective therapy. The MEK inhibitor binimetinib shows an overall response rate of 15% in patients with NRAS-mutant melanoma, providing a backbone for combination strategies. Our previous studies demonstrated that in NRAS-mutant melanoma, the antitumor activity of the MEK inhibitor binimetinib was significantly potentiated by the BRAFV600E/K inhibitor encorafenib through the induction of ER stress, leading to melanoma cell death by apoptotic mechanisms. Encorafenib combined with binimetinib was well tolerated in a phase III trial showing potent antitumor activity in BRAF-mutant melanoma, making a rapid evaluation in NRAS-mutant melanoma imminently feasible. These data provide a mechanistic rationale for the evaluation of binimetinib combined with encorafenib in preclinical and clinical studies on NRAS-mutant metastatic melanoma. The combination of BRAFi plus MEKi was tested in a monolayer culture of patient-derived cell lines and in corresponding patient-derived tissue slice cultures of NRAS-mutant melanoma. To investigate the treatment in vivo, NSG (NOD. Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice were subcutaneously injected with three different BRAF wild-type melanoma models harboring oncogenic NRAS mutations and treated orally with encorafenib (6 mg/kg body weight, daily) with or without binimetinib (8 mg/kg body weight, twice daily). In parallel, an individual healing attempt was carried out by treating one patient with an NRAS-mutated tumor. Encorafenib was able to enhance the inhibitory effect on cell growth of binimetinib only in the cell line SKMel147 in vitro. It failed to enhance the apoptotic effect found in two other NRAS-mutated cell lines. Encorafenib led to a hyperactivation of ERK which could be reduced with the combinational treatment. In two of the three patient-derived tissue slice culture models of NRAS-mutant melanomas, a slight tendency of a combinatorial effect was seen which was not significant. Encorafenib showed a slight induction of the ER stress genes ATF4, CHOP, and NUPR1. The combinational treatment was able to enhance this effect, but not significantly. In the mouse model, the combination therapy of encorafenib with binimetinib resulted in reduced tumor growth compared to the control and encorafenib groups; however, the best effect in terms of tumor growth inhibition was measured in the binimetinib therapy group. The therapy showed no effect in an individual healing attempt for a patient suffering from metastatic, therapy-refractory NRAS-mutated melanoma. In in vitro and ex vivo settings, the combination therapy was observed to elicit a response; however, it did not amplify the efficacy observed with binimetinib alone, whereas in a patient, the combinational treatment remained ineffective. The preclinical in vivo data showed no increased combinatorial effect. However, the in vivo effect of binimetinib as monotherapy was unexpectedly high in the tested regimen. Nevertheless, binimetinib proved to be advantageous in the treatment of melanoma in vivo and led to high rates of apoptosis in vitro; hence, it still seems to be a good base for combination with other substances in the treatment of patients with NRAS-mutant melanoma.

Targeting ribosome biogenesis reinforces ERK-dependent senescence in pancreatic cancer

ABSTRACTPancreatic adenocarcinomas (PDAC) often possess mutations in K-Ras that stimulate the ERK pathway. Aberrantly high ERK activation triggers oncogene-induced senescence, which halts tumor progression. Here we report that low-grade pancreatic intraepithelial neoplasia displays very high levels of phospho-ERK consistent with a senescence response. However, advanced lesions that have circumvented the senescence barrier exhibit lower phospho-ERK levels. Restoring ERK hyperactivation in PDAC using activated RAF leads to ERK-dependent growth arrest with senescence biomarkers. ERK-dependent senescence in PDAC was characterized by a nucleolar stress response including a selective depletion of nucleolar phosphoproteins and intranucleolar foci containing RNA polymerase I designated as senescence-associated nucleolar foci (SANF). Accordingly, combining ribosome biogenesis inhibitors with ERK hyperactivation reinforced the senescence response in PDAC cells. Notably, comparable mechanisms were observed upon treatment with the platinum-based chemotherapy regimen FOLFIRINOX, currently a first-line treatment option for PDAC. We thus suggest that drugs targeting ribosome biogenesis can improve the senescence anticancer response in pancreatic cancer.

A phase 1/2 study of CFT1946, a novel, bifunctional degradation activating compound (BIDAC) degrader, of mutant BRAF V600 as monotherapy and in combination with trametinib, in mutant BRAF V600 solid tumors.

TPS3163 Background: BRAF is a protein kinase that acts as a signal transducer in the MAPK pathway. Mutant BRAF V600 is capable of uncontrolled signaling and is constitutively active and signals as a monomer, leading to hyperactivation of MEK, ERK, and dysregulation of cellular proliferation. Mutant BRAF V600 is a clinically-validated target in a variety of cancers including melanoma, colorectal cancer (CRC), non-small cell lung cancer (NSCLC), and anaplastic thyroid carcinoma (ATC). Currently approved BRAF inhibitors (BRAFi) result in paradoxical RAF activation as the mutant BRAF protein is still able to dimerize with WT BRAF, resulting in a dimeric signaling complex. CFT1946 is a novel, orally bioavailable BiDAC degrader that inhibits and degrades mutant BRAF V600 protein. Distinct from approved BRAFi, CFT1946 avoids paradoxical RAF activation as the degraded BRAF V600 mutant protein can no longer incorporate into a dimeric signaling complex. CFT1946 is selective for the mutant protein and spares WT BRAF V600. CFT1946 has demonstrated preclinical activity in BRAF V600 mutant in vitro and in vivo models, including models resistant to BRAFi. These preclinical data provide rationale for a first-in-human (FIH) study to evaluate CFT1946 in BRAF V600 mutant solid tumors. Methods: This is a FIH, open-label, multicenter, Phase 1/2 clinical trial to characterize the initial safety, tolerability, and preliminary efficacy of CFT1946 and CFT1946+trametinib in BRAF V600 mutant melanoma, NSCLC, CRC, ATC, and other non-CNS BRAF V600 mutant solid tumors. This study includes 2 phases: dose escalation Phase 1 comprising CFT1946 (Arm A) with a starting oral dose of 20 mg twice daily and CFT1946+trametinib (Arm B) as well as dose expansion Phase 2 comprising CFT1946 (Arm A1) and CFT1946+trametinib (Arms B1 and B2). Arm A1 and B1 will include BRAF V600 mutant melanoma and NSCLC with prior BRAFi therapy while Arm B2 will enroll BRAF V600 mutant NSCLC who are BRAFi-naïve. Phase 2 will be initiated once the RP2D has been identified. Eligible subjects are ≥18yo with documented BRAF V600 mutant cancers who have received ≥1 prior therapy. CFT1946 will be administered orally in 28-day cycles until disease progression or intolerable toxicity. Primary objectives include safety, tolerability, RP2D and preliminary antitumor activity of CFT1946 monotherapy and in combination with trametinib. Secondary objectives include assessment of pharmacokinetics and pharmacodynamics. Key inclusion criteria include documented evidence of a BRAF V600 mutation obtained from tumor tissue or liquid biopsy; at least 1 prior line of standard-of-care therapy for locally advanced or metastatic disease. The study is anticipated to enroll 101-122 patients across 11 participating US and European sites and is currently open to enrollment (NCT05668585). Clinical trial information: NCT05668585 .

Abstract A010: Using oncogenic pathway agonism to sensitize RAS-mutant cancers to immunotherapy

Abstract Overall, some 30% of human cancers are driven by mutant RAS proteins, which have proven relatively difficult to target. While immunotherapy is effective for NRAS-mutant melanoma, no options exist for resistant disease, and mutant KRAS-driven lung and pancreatic carcinomas are much less responsive overall. Oncogenic pathways are often key targets for inhibition. However, pathway agonism has not been systematically explored as a therapeutic approach. BRAF-mutant melanomas respond to BRAF inhibitors (BRAFi) due to decreased ERK signaling. They can recover signaling by acquiring activating RAS/MEK mutations, but exhibit decreased proliferation in the absence of inhibitor, suggesting that supraphysiologic ERK signaling also compromises fitness. In line with this, we observed that increasing ERK hyperactivation in RAS-mutant cancers, particularly in those with a high ERK activity, might elevate ERK signaling to a degree to induce senescence and also create an inflammatory tumor microenvironment. We have shown that a majority of RAS-mutant cancer cell lines we tested undergo senescence-like arrest following exposure to all FDA-approved BRAF inhibitors at clinically relevant doses. Simultaneous MEK or ERK inhibition allows the cells to recover proliferation, and a BRAFi which does not induce ERK activation fails to have any effect, confirming that ERK hyperactivation is the key driver of this response. Importantly, these findings generalize across NRAS-mutant and KRAS-mutant cancer cell lines including in fresh human pancreatic adenocarcinoma explants. Finally, our data in two novel genomically-characterized, immunocompetent models of NRAS-mutant melanoma and KRAS-mutant pancreatic adenocarcinoma support our in-vitro findings. Both RAS-mutant murine models demonstrate that anti-PD1 therapy is substantially more efficacious when mice are additionally treated with BRAFi. Molecular profiling of the tumor microenvironment reveals the expression of cytokines related to senescence and marked infiltration of activated CD8+ T-cells. In order to implement more potentially effective immunotherapies, we have analyzed the expression of different immune-checkpoint molecules and we have observed a high expression of LAG3 and TIM3 in the melanoma NRAS-mutant model. We will further analyze the effect of those checkpoints in combination with BRAFi to potentiate the anti-tumoral responses and tumor regression. We propose that oncogenic pathway agonism is a novel, effective, and untested strategy to induce proliferation arrest and sensitization to immunotherapy. The impact of this data is in the ability to broadly sensitize RAS-mutant cancers to immunotherapy in the settings of de-novo and acquired resistance. The direct connection between a tumor cell signaling vulnerability to immunotherapy response is a major point of novelty and provides a clear molecular rationale for pursuing combined targeted and immune-based therapy. Citation Format: Alvaro de Mingo Pulido, Charles Adelmann, Brittney Sell, Karol Prieto, Kimberly Nguyen, Cynthia Dixey, Ivannie Ortiz-Rivera, Marco Napoli, Elsa Flores, Jason Fleming, Karen Mann, Christin Burd, Kenneth Y. Tsai. Using oncogenic pathway agonism to sensitize RAS-mutant cancers to immunotherapy [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A010.

MEK inhibitor resistance in lung adenocarcinoma is associated with addiction to sustained ERK suppression

MEK inhibitors (MEKi) have limited efficacy in KRAS mutant lung adenocarcinoma (LUAD) patients, and this is attributed to both intrinsic and adaptive mechanisms of drug resistance. While many studies have focused on the former, there remains a dearth of data regarding acquired resistance to MEKi in LUAD. We established trametinib-resistant KRAS mutant LUAD cells through dose escalation and performed targeted MSK-IMPACT sequencing to identify drivers of MEKi resistance. Comparing resistant cells to their sensitive counterparts revealed alteration of genes associated with trametinib response. We describe a state of “drug addiction” in resistant cases where cells are dependent on continuous culture in trametinib for survival. We show that dependence on ERK2 suppression underlies this phenomenon and that trametinib removal hyperactivates ERK, resulting in ER stress and apoptosis. Amplification of KRASG12C occurs in drug-addicted cells and blocking mutant-specific activity with AMG 510 rescues the lethality associated with trametinib withdrawal. Furthermore, we show that increased KRASG12C expression is lethal to other KRAS mutant LUAD cells, consequential to ERK hyperactivation. Our study determines the drug-addicted phenotype in lung cancer is associated with KRAS amplification and demonstrates that toxic acquired genetic changes can develop de novo in the background of MAPK suppression with MEK inhibitors. We suggest that the presence of mutant KRAS amplification in patients may identify those that may benefit from a “drug holiday” to circumvent drug resistance. These findings demonstrate the toxic potential of hyperactive ERK signaling and highlight potential therapeutic opportunities in patients bearing KRAS mutations.

Pharmacologic Inhibition of DYRK1A Results in Hyperactivation and Hyperphosphorylation of MYC and ERK Rendering KMT2A-R ALL Cells Sensitive to BCL2 Inhibition

Background: B-cell acute lymphoblastic leukemia (ALL) harboring KMT2A-rearrangements (KMT2A-R) is a high-risk ALL subtype with significant relapse risk and poor overall survival across the age spectrum. While new antibody-based and cellular immunotherapies have revolutionized the therapeutic paradigm for many patients with relapsed ALL, application of these therapies has proven particularly challenging to date in infants given their immune system immaturity and in older adults prone to high-grade immunotherapy toxicities. Unlike other ALL subtypes, KMT2A-R ALL has an extremely low frequency of co-mutations, which makes it particularly difficult to find new treatment options. To identify targetable kinases in KMT2A-R ALL, we performed a domain-specific kinome-wide CRISPR screen and identified the serine/threonine kinase DYRK1A as required for the survival of KMT2A-R ALL. Importantly, DYRK1A appears to be non-essential for normal tissues per the Cancer Dependency MAP, suggesting potential for a therapeutic window via DYRK1A targeting. Results:In vitro treatment studies using three different menin inhibitors (MI-2-2 / MI-503 / VTP50469) to disrupt the transcriptional activity of the KMT2A-R complex resulted in the downregulation of DYRK1A at the RNA and the protein level. We further analyzed ChIP-Seq data and identified that oncogenic KMT2A-R directly binds to the DYRK1A promoter, indicating that DYRK1A is directly regulated by the oncogenic KMT2A fusion. Interestingly, the DYRK1A RNA and protein levels were elevated in KMT2A-R ALL cell lines and patient-derived xenograft (PDX) samples compared to non-KMT2A-R ALL cases. To test the consequence of direct pharmacologic DYRK1A inhibition on cell proliferation and apoptosis, we treated KMT2A-R ALL cells with the DYRK1A inhibitors EHT1610 and harmine. Interestingly, we observed that pharmacologic inhibition of DYRK1A with both drugs did not induce apoptosis but induced potent leukemic cell growth inhibition in vitro and in vivo. In preliminary studies, we further observed that pharmacologic inhibition of DYRK1A or genetic deletion resulted in MYC upregulation and hyperphosphorylation of ERK. It has been demonstrated that premature signaling activation in developing B cells induces cell cycle arrest as a protective mechanism to prevent the development of autoimmune diseases. We hypothesized that DYRK1A inhibitor-induced ERK hyperactivation and consequent cell cycle arrest remains a conserved mechanism in KMT2A-R ALL. Supporting our hypothesis, combining DYRK1A inhibitors with the MEK inhibitor trametinib antagonistically rescued KMT2A-R ALL cell proliferation, indicating that ERK hyperactivation is the main driver of DYRK1A inhibitor mediated cell cycle arrest. Given that DYRK1A inhibition does not induce apoptosis and cells restart to proliferate after DYRK1A inhibitor withdrawal, we concluded that a DYRK1A monotherapy may not be an ideal new treatment option. Interestingly, it has been reported that increased MYC activity induces the accumulation of BIM in Burkitt's Lymphoma. BIM is a pro-apoptotic molecule that is inactive when bound by the anti-apoptotic molecule BCL2. Given that DYRK1A inhibition results in MYC upregulation, we performed a new Western blot analysis and validated that EHT1610 and harmine both induce BIM overexpression in KMT2A-R ALL cell lines. To test if combined DYRK1A and BCL2 inhibition will lead to enhanced KMT2A-R ALL apoptosis, we treated four KMT2A-R ALL cell lines with increasing concentrations of EHT1610 and the BCL2 inhibitor venetoclax. Strikingly, combining both drugs synergistically killed KMT2A-R ALL cells in vitro. More importantly, we validated the anti-leukemic effect of harmine in combination with venetoclax in mice injected with a KMT2A-R PDX case. Conclusion: Our results validate DYRK1A as an important molecule to regulate cell proliferation via inhibition of MYC and ERK. Targeting DYRK1A results in the accumulation of BIM, which renders the cells sensitive to BCL2 inhibition via venetoclax. While further in vivo studies are needed, we predict that combining DYRK1A inhibition with venetoclax may be a novel precision medicine strategy for the treatment of KMT2A-R ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P324: PHARMACOLOGIC INHIBITION OF DYRK1A RENDERS HIGH-RISK KMT2A-R ALL SENSITIVE TO VENETOCLAX

Background:KMT2A-rearranged (R) ALL is a high-risk disease with a frequency of 70% in infants and 10% in children and adults with ALL and is associated with chemoresistance, relapse, and poor survival. Current intensive multiagent chemotherapy regimens induce significant side effects, yet fail to cure many patients, demonstrating continued need for novel therapeutic approaches. Aims: Determine if pharmacologic DYRK1A inhibition may be a novel treatment strategy for patients with KMT2A-R ALL. Methods: To identify novel targets in KMT2A-R leukemia, we performed a domain-specific kinome-wide CRISPR screen and identified multiple kinases required for cell growth. We focused on DYRK1A as it met the following three criteria: 1) Growth inhibition upon kinase targeting was greater in KMT2A-R leukemic cells than in non-KMT2A-R cells, 2) DYRK1A was not found to be common essential gene assessed through the Cancer Dependency Map, 3) Small molecule inhibitors are available. Results: We analyzed multiple ChIP-Seq experiments and identified that KMT2A-fusions directly bind to the DYRK1A promoter. Our RT-PCR and Western blot analyses demonstrate that KMT2A-R ALL cells treated with a menin inhibitor to disrupt the transcriptional activity of the KMT2A-R complex, downregulate DYRK1A, indicating direct regulation of DYRK1A by the KMT2A-fusion. We further observed that pharmacologic inhibition of DYRK1A with EHT1610 induced leukemic cell growth inhibition in vitro and in vivo, demonstrating that DYRK1A could be a new therapeutic target in KMT2A-R ALL cells. To further elucidate the mechanism of DYRK1A function, we treated several KMT2A-R ALL cell lines in vitro with EHT1610, which surprisingly resulted in the upregulation of MYC and hyperphosphorylation of the RAS/MAPK target ERK. Given that ERK hyperactivation stops B cell proliferation during early B cell development to allow them to rearrange their B cell receptor, we hypothesized that cell cycle inhibition upon ERK hyperactivation remains as a conserved mechanism of cell cycle regulation in KMT2A-R ALL. Strikingly, combining DYRK1A inhibition with the MEK inhibitor trametinib antagonistically rescued KMT2A-R ALL cell proliferation, indicating that ERK hyperactivation is the main driver of DYRK1A inhibitor mediated cell cycle arrest. Given that DYRK1A inhibitor does not induce apoptosis and cells restart cell proliferation after EHT1610 withdrawal we concluded that a DYRK1A monotherapy may not be an ideal new treatment option. However, it has been reported that increased MYC activity induces the accumulation of BIM in Burkitt’s Lymphoma. Given the increased expression of MYC following DYRK1A inhibition we performed a new Western blot analysis and validated increased expression of BIM in our KMT2A-R ALL cell lines after EHT1610 treatment. To test if targeting the interaction of BIM with BCL2 will induce an apoptotic effect when combined with EHT1610, we treated four KMT2A-R ALL cell lines with increasing concentrations of EHT1610 and the BCL2 inhibitor venetoclax. Strikingly, the combination of DYRK1A inhibition with BCL2 inhibition synergistically killed KMT2A-R ALL cells and significantly reduced the leukemia burden in vivo. Image:Summary/Conclusion: Our results validate DYRK1A as an important molecule to regulate cell proliferation via inhibition of MYC and ERK. Targeting DYRK1A results in the accumulation of BIM, which renders the cells sensitive to BCL2 inhibition via venetoclax. While further in vivo validation is needed, we predict that combining DYRK1A inhibition with venetoclax may be a novel precision medicine strategy for the treatment of KMT2A-R ALL.

DUSP4 protects BRAF- and NRAS-mutant melanoma from oncogene overdose through modulation of MITF.

MAPK inhibitors (MAPKi) remain an important component of the standard of care for metastatic melanoma. However, acquired resistance to these drugs limits their therapeutic benefit. Tumor cells can become refractory to MAPKi by reactivation of ERK. When this happens, tumors often become sensitive to drug withdrawal. This drug addiction phenotype results from the hyperactivation of the oncogenic pathway, a phenomenon commonly referred to as oncogene overdose. Several feedback mechanisms are involved in regulating ERK signaling. However, the genes that serve as gatekeepers of oncogene overdose in mutant melanoma remain unknown. Here, we demonstrate that depletion of the ERK phosphatase, DUSP4, leads to toxic levels of MAPK activation in both drug-naive and drug-resistant mutant melanoma cells. Importantly, ERK hyperactivation is associated with down-regulation of lineage-defining genes including MITF Our results offer an alternative therapeutic strategy to treat mutant melanoma patients with acquired MAPKi resistance and those unable to tolerate MAPKi.

A kinase translocation reporter reveals real-time dynamics of ERK activity in Drosophila.

ABSTRACTExtracellular signal-regulated kinase (ERK) lies downstream of a core signalling cascade that controls all aspects of development and adult homeostasis. Recent developments have led to new tools to image and manipulate the pathway. However, visualising ERK activity in vivo with high temporal resolution remains a challenge in Drosophila. We adapted a kinase translocation reporter (KTR) for use in Drosophila, which shuttles out of the nucleus when phosphorylated by ERK. We show that ERK-KTR faithfully reports endogenous ERK signalling activity in developing and adult tissues, and that it responds to genetic perturbations upstream of ERK. Using ERK-KTR in time-lapse imaging, we made two novel observations: firstly, sustained hyperactivation of ERK by expression of dominant-active epidermal growth factor receptor raised the overall level but did not alter the kinetics of ERK activity; secondly, the direction of migration of retinal basal glia correlated with their ERK activity levels, suggesting an explanation for the heterogeneity in ERK activity observed in fixed tissue. Our results show that KTR technology can be applied in Drosophila to monitor ERK activity in real-time and suggest that this modular tool can be further adapted to study other kinases.

Mechanisms underlying melanoma invasion as a consequence of MLK3 loss

Invasive melanoma is an aggressive form of skin cancer with high incidence of mortality. The process of tumor invasion is a crucial primary step in the metastatic cascade, yet the mechanisms involved are still under investigation. Here we document a critical role for MLK3 (MAP3K11) in the regulation of melanoma cell invasion. We report the unexpected finding that cellular loss of MLK3 in melanoma cells promotes cell invasion. Cellular depletion of MLK3 expression results in the hyperactivation of ERK, which is linked to the formation of a BRAF/Hsp90/Cdc37 protein complex. ERK hyperactivation leads to enhanced phosphorylation and inactivation of GSK3β and the stabilization of c-Jun and JNK activity. Blocking of ERK and JNK signaling as well as Hsp90 activity downstream of MLK3-silencing significantly reduces melanoma invasion. Furthermore, ERK activation in the aforementioned context is coupled to MT1-MMP transcription as well as the TOM1L1-dependent localization of the membrane protease to invadopodia at the invasive front. These studies provide critical insight into the mechanisms that couple MLK3 loss with BRAF hyperactivation and its consequence on melanoma invasion.

Abstract OT2-19-08: Phase IB/II trial of palbociclib and binimetinib in advanced triple-negative breast cancer with hyperactivation of ERK and/or CDK4/6

Abstract Background: Novel effective and safe therapies are required for advanced TNBC after progression to standard-of-care first line treatment with anti-PD-1/L1 + chemotherapy. We have found that the most aggressive TNBC variants are driven by a heterogeneous set of genetic aberrations that converge in the increased activity of 6 kinases: KIT, PNKP, PRKCE, P70S6K, ERK and CDK6 (Nat Commun; 9:3501-18). Although the inhibition of each kinase in monotherapy yielded little efficacy in preclinical models, the combinations targeting pairs of the former kinases led to therapeutic synergy in most cases. The combined inhibition of CDK6 and ERK led to 5-fold increase in overall survival in preclinical TNBC models (PDXs and spontaneous murine cancer models). The greatest activity was observed in models with increased activity of either CDK6 or ERK. Thus, we aimed to test the safety and preliminary efficacy of combined ERK and CDK6 inhibition with binimetinib and palbociclib in women with advanced TNBC with hyperactivation of ERK and/or CDK6. The combination has been preliminary tested in lung cancer, where a phase I dose-escalation trial established the RP2D in binimetinib 45 mg/BID plus Palbociclib 125 mg daily 21/7. Trial design: Single-arm, prospective, multicentric, open-label, phase IB/II trial with intra-patient dose-escalation. Patients candidate for pre-screening will have determined the activity of ERK and CDK6 with an in-house developed assay and acquisition algorithm at the central lab. Those testing positive for either marker will undergo full screening. Patients will start treatment with continuous oral binimetinib at 45 mg/BID and palbociclib 100 mg/day from days 1 to 21, in 28-day cycles. Patients experiencing ≤ grade 1 tolerable side effects as the greatest toxicity will be allowed to escalate to palbociclib to 125 in cycle 2. Fresh biopsies will be harvested at baseline and disease progression, in order to establish patient-derived organoids (PDOs) and perform WES. PBMCs will be harvested at baseline, +24 hours, and at the beginning of cycle 2, to study changes in the immunophenotype. RECIST 1.1 and NCI CTC AE V 5.0 criteria will be used for assessing disease control (q8 weeks) and toxicity. Eligibility criteria: Pre-screening: Women >18 year old diagnosed of advanced, non-curable TNBC; 2) who have received a minimum of 1 treatment line including immunotherapy; 3) no more than 2 treatment lines for advanced disease; 4) Adequate organ function and recovery from previous toxicity to < tolerable G2. Full screening: 5) Demonstration of hyper-activation of CDK6 and/or ERK in the tumor sample; 6) PD to the previous treatment regimen within the last 28 days. Specific aims: Primary: 1) To determine the progression-free survival time of the combination of binimetinib and palbociclib in TNBC patients with hyperactivation of ERK and/or CDK6 in second/third line 2) To assess the safety and tolerability of the former combination Secondary: 1) 6-month PFS rate, response rate, and overall survival of the combination 2) To study biomarkers of sensitivity and primary and acquired resistance to the combination taking advantage of PDOs 3) To determine the immunodynamics of the combination. Statistical methods: The null hypothesis is that the median PFS time for second/third line TNBC with best physician’s choice is 1.7 months (NEJM; 384:1529-41, 2021). With alpha and beta errors of 0.05 and 0.2, the minimum number of patients to demonstrate a 30% improvement in PFS to 2.5 months is 25. Preliminary data suggest that approximately 40% of the patients show hyper-activation of CDK6 and/or ERK; thus, and assuming a methodological failure of 10%, the minimum number of patients to screen is 69. Present accrual/target accrual: 27 screened of 69 planed; 11 accrued of 25 planned Citation Format: Luis Manso, Alfonso Cortes, Juan M Cejalvo, Serafin Morales, Jose A García Saenz, Ramon Colomer, Rodrigo Sanchez-Bayona, Jorge Silva, Juan A Guerra, Diego Malon, Silvana Mouron, Eduardo Caleiras, Miguel Quintela-Fandino. Phase IB/II trial of palbociclib and binimetinib in advanced triple-negative breast cancer with hyperactivation of ERK and/or CDK4/6 [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-19-08.

Patient-Derived Ovarian Cancer Spheroids Rely on PI3K-AKT Signaling Addiction for Cancer Stemness and Chemoresistance.

Simple SummaryEpithelial ovarian cancer (EOC) is the most fatal gynecological cancer with poor survival rates and high mortality. EOC patients respond to standard platinum-based chemotherapy in the beginning, but relapse often due to chemoresistance. Ovarian cancer cells disseminate from the ovarian tumors and spread within the abdomen, where ascites fluid supports the growth and transition. Malignant ascites is present in a third of patients at diagnosis and is considered as a major source of chemoresistance, recurrence, poor survival, and mortality. Malignant ascites is a complex fluid that contains a pro-tumorigenic environment with disseminated cancer cells in 3D spheroids form. In this study, we established an ovarian cancer cell line and identified that 3D spheroids develop from the 2D monolayer, and the platinum-resistant phenotype develops due to the aberrant PI3K-AKT signaling in tumor cells. Furthermore, when we used a combinatorial approach of cisplatin with LY-294002 (a PI3K-AKT dual kinase inhibitor) to treat the cisplatin version of both MCW-OV-SL-3 and A-2780 cell lines, it prevented the 3D spheroid formation ability and also sensitized the cells for cisplatin. In brief, our results provided evidence to advance therapeutic approaches to treat cisplatin resistance in ovarian cancer patients.Ovarian cancer is the most lethal gynecological malignancy among women worldwide and is characterized by aggressiveness, cancer stemness, and frequent relapse due to resistance to platinum-based therapy. Ovarian cancer cells metastasize through ascites fluid as 3D spheroids which are more resistant to apoptosis and chemotherapeutic agents. However, the precise mechanism as an oncogenic addiction that makes 3D spheroids resistant to apoptosis and chemotherapeutic agents is not understood. To study the signaling addiction mechanism that occurs during cancer progression in patients, we developed an endometrioid subtype ovarian cancer cell line named ‘MCW-OV-SL-3’ from the ovary of a 70-year-old patient with stage 1A endometrioid adenocarcinoma of the ovary. We found that the cell line MCW-OV-SL-3 exhibits interstitial duplication of 1q (q21–q42), where this duplication resulted in high expression of the PIK3C2B gene and aberrant activation of PI3K-AKT-ERK signaling. Using short tandem repeat (STR) analysis, we demonstrated that the cell line exhibits a unique genetic identity compared to existing ovarian cancer cell lines. Notably, the MCW-OV-SL-3 cell line was able to form 3D spheroids spontaneously, which is an inherent property of tumor cells when plated on cell culture dishes. Importantly, the tumor spheroids derived from the MCW-OV-SL-3 cell line expressed high levels of c-Kit, PROM1, ZEB1, SNAI, VIM, and Twist1 compared to 2D monolayer cells. We also observed that the hyperactivation of ERK and PI3K/AKT signaling in these cancer cells resulted in resistance to cisplatin. In summary, the MCW-OV-SL3 endometrioid cell line is an excellent model to study the mechanism of cancer stemness and chemoresistance in endometrioid ovarian cancer.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Pharmacologic Inhibition of DYRK1A Results in Hyperactivation and Hyperphosphorylation of MYC and ERK Rendering KMT2A-R ALL Cells Sensitive to BCL2 Inhibition

Background: KMT2A-rearranged (R) ALL is a high-risk disease with a frequency of 75% in infants and 10% in children and adults with ALL and is associated with chemoresistance, relapse, and poor survival. Current intensive multiagent chemotherapy regimens induce significant side effects, yet fail to cure many patients, demonstrating continued need for novel therapeutic approaches. We performed a kinome-wide CRISPR screen and identified that DYRK1A is specifically required for the survival of KMT2A-R ALL cell. DYRK1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and has been reported as negatively regulator of cell proliferation.Results: We performed a kinome-wide CRISPR screen in human ALL cell lines and PDX models and identified DYRK1A as a novel target in KMT2A-R ALL. DYRK1A is a serine-threonine kinase with a proposed, but poorly defined role in cell cycle regulation. We performed a meta-analysis of multiple ChIP-Seq experiments and identified that oncogenic KMT2A fusions directly bind to the DYRK1A promoter. Our RT-PCR and Western blot analyses of KMT2A-R ALL cells treated with a menin inhibitor (MI-503) to disrupt the transcriptional activity of the KMT2A-R complex resulted in the downregulation of DYRK1A, indicating that DYRK1A is directly regulated by the KMT2A fusion complex. We further observed that pharmacologic inhibition of DYRK1A with EHT1610 induced potent leukemic cell growth inhibition in vitro and in vivo, demonstrating that DYRK1A could be a new therapeutic target in KMT2A-R ALL cells. To further elucidate the mechanism of DYRK1A function, we treated several KMT2A-R ALL cell lines in vitro with EHT1610, which surprisingly resulted in the upregulation of MYC and hyperphosphorylation of the RAS/MAPK target ERK. Given that ERK hyperactivation stops B cell proliferation during early B cell development to allow them to rearrange their B cell receptor, we hypothesized that cell cycle inhibition upon ERK hyperactivation remains as a conserved mechanism of cell cycle regulation in KMT2A-R ALL. Strikingly, combining DYRK1A inhibition with the MEK inhibitor trametinib antagonistically rescued KMT2A-R ALL cell proliferation, indicating that ERK hyperactivation is the main driver of DYRK1A inhibitor mediated cell cycle arrest. Given that DYRK1A inhibitor does not induce apoptosis and cells restart cell proliferation after EHT1610 withdrawal we concluded that a DYRK1A monotherapy may not be an ideal new treatment option. However, it has been reported that increased MYC activity induces the accumulation of BIM in Burkitt's Lymphoma. Given the increased expression of MYC following DYRK1A inhibition we performed a new Western blot analysis and validated increased expression of BIM in our KMT2A-R ALL cell lines after EHT1610 treatment. To test if targeting the interaction of BIM with BCL2 will induce an apoptotic effect when combined with EHT1610, we treated four KMT2A-R ALL cell lines with increasing concentrations of EHT1610 and the BCL2 inhibitor venetoclax. Strikingly, the combination of DYRK1A inhibition with BCL2 inhibition synergistically killed KMT2A-R ALL cells.Conclusion: Our results validate DYRK1A as an important molecule to regulate cell proliferation via inhibition of MYC and ERK. Targeting DYRK1A results in the accumulation of BIM, which renders the cells sensitive to BCL2 inhibition via venetoclax. While further in vivo studies are needed, we predict that combining DYRK1A inhibition with venetoclax may be a novel precision medicine strategy for the treatment of KMT2A-R ALL. [Display omitted] DisclosuresCrispino: Forma Therapeutics: Research Funding; Scholar Rock: Research Funding; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy. Tasian: Aleta Biotherapeutics: Consultancy; Gilead Sciences: Research Funding; Kura Oncology: Consultancy; Incyte Corporation: Research Funding. Carroll: Incyte Pharmaceuticals: Research Funding; Janssen Pharmaceutical: Consultancy.

NKX2-1 controls lung cancer progression by inducing DUSP6 to dampen ERK activity.

SummaryThe RAS→RAF→MEK→ERK pathway is hyperactivated in the majority of human lung adenocarcinoma (LUAD). However, the initial activating mutations induce homeostatic feedback mechanisms that limit ERK activity. How ERK activation reaches the tumor-promoting levels that overcome the feedback and drive malignant progression is unclear. We show here that the lung lineage transcription factor NKX2-1 suppresses ERK activity. In human tissue samples and cell lines, xenografts, and genetic mouse models, NKX2-1 induces the ERK phosphatase DUSP6, which inactivates ERK. In tumor cells from late-stage LUAD with silenced NKX2-1, re-introduction of NKX2-1 induces DUSP6 and inhibits tumor growth and metastasis. We show that DUSP6 is necessary for NKX2-1-mediated inhibition of tumor progression in vivo and DUSP6 expression is sufficient to inhibit RAS-driven LUAD. Our results indicate that NKX2-1 silencing, and thereby DUSP6 downregulation, is a mechanism by which early LUAD can unleash ERK hyperactivation for tumor progression.

BCL6 Enables RAS-Driven Pre-B Cell Transformation in Acute Lymphoblastic Leukemia

Background and Hypothesis: The transcriptional repressor and proto-oncogene BCL6 was previously identified to be a factor that bypasses oncogene-induced senescence (OIS) caused by activation of oncogenic RAS (Shvarts A. et al. 2002). Recently, we discovered a novel BCL6-dependent mechanism of drug resistance in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL; Duy et al., 2011). Given that activating genetic lesions in the RAS pathway are found in ~50% of high-risk childhood pre-B ALL at diagnosis (Zhang et al., 2012), we tested the concept that BCL6 may facilitate RAS-mediated transformation of pre-B cells by suppressing the OIS-program.Results: To examine the consequences of acute activation of RAS-ERK signaling on Bcl6 expression, we engineered murine pre-B cells to carry a doxycycline (Dox)-inducible TetOn- NRASG12D vector system. Following induction with Dox, strong upregulation of Bcl6 expression was observed at both the mRNA and protein levels, which were abrogated upon treatment with a MEK inhibitor (PD325901). These findings suggest that increased Bcl6 levels were a consequence of ERK activation. Similarly, inducible expression of BRAF V600E mutant resulted in elevated levels of Bcl6 in parallel with increased phosphorylation of ERK1/2- T202/Y204. Furthermore, studying a diagnostic (KRAS wild-type) and a relapsed (KRASG12V) sample from one pre-B ALL patient revealed hyperactivation of ERK in association with increased BCL6 levels in KRASG12V relapsed ALL cells.Previously, we showed that BCL6 expression is repressed by STAT5 in pre-B ALL (Duy C. et al. 2011). Interestingly, while overexpression of oncogenic NRASG12D resulted in hyperactivation of ERK in pre-B cells, phosphorylation of STAT5-Y694 was drastically inhibited. Reverse Phase Protein Array (RPPA) measurements revealed that phosphorylation levels of MEK-S217/221 and ERK1/2-T202/Y204 were inversely correlated with phosphorylation of STAT5-Y694 in newly diagnosed cases of adult ALL (MDACC, 1983-2007). In addition, genetic lesions causing activation of the RAS-ERK and STAT5 pathways were found to be mutually exclusive in the majority of high-risk childhood pre-B ALL cases studied (COG P9906). Mechanistically, expression of oncogenic NRASG12D resulted in activation of PTPN6 (SHP1), an inhibitory protein tyrosine phosphatase. Loss of Ptpn6 function resulted in elevated levels of phospho-STAT5-Y694 and suppressed imatinib-mediated induction of Bcl6 expression in BCR-ABL1 -driven pre-B ALL. Taken together, our findings suggest that oncogenic RAS induces BCL6 expression through PTPN6-mediated dephosphorylation of STAT5.Our observations that RAS-ERK signaling induces BCL6 expression prompted us to examine the role of BCL6 in RAS-mediated malignant transformation of pre-B cells. Activation of oncogenic RAS increased expression of cell cycle checkpoint regulators CDKN1A (p21) and CDKN1B (p27). BCL6 has previously been shown to mediate transcriptional repression of CDKN1A and CDKN1B (Duy C. et al. 2011). Notably, ChIP-seq analysis of patient-derived KRASG12V pre-B ALL cells revealed binding of BCL6 across the loci of cell cycle checkpoint regulators CDKN1A, CDKN1B, CDKN2D and TP53BP1, as well as pro-apoptotic molecules BIM and BMF . Consistent with these findings, Cre-mediated deletion of Bcl6 in pre-B cells expressing NRASG12D led to depletion of cells in competitive-growth assays and impaired the ability to form colonies in semi-solid methylcellulose. Moreover, expression of NRASG12Din Bcl6+/+ pre-B cells resulted in transformation and fatal leukemia in transplant recipient mice. In contrast, Bcl6-/- pre-B cells transduced with NRASG12D failed to initiate fatal disease in transplant recipients.Conclusions: Our findings suggest that activation of oncogenic RAS enables the switch from STAT5-driven signaling to activation of BCL6, and that RAS-induced dephosphorylation of STAT5 is mediated by PTPN6. Furthermore, we demonstrated that BCL6 is required for RAS -mediated transformation of pre-B cells, suggesting that inhibition of BCL6 may provide a new therapeutic avenue. DisclosuresHurtz:Incyte: Research Funding.

NKX2‐1 controls lung cancer progression by inducing DUSP6 to dampen ERK activity

Lung cancer remains a leading cause of cancer death, with unclear mechanisms driving the transition to aggressive cancer with poor prognosis. The RAS/RAF/MEK/ERK pathway is hyper-activated in ~50% of human lung adenocarcinoma (LUAD). An initial activating mutation induces homeostatic feedback mechanisms that limit ERK activity. Additional, undefined genetic hits overcome the feedback, leading to high ERK activity that drives malignant progression. At detection, the majority of LUADs express the homeobox transcription factor NKX2-1, which also limits malignant progression. NKX2-1 constrains LUAD, in part, by maintaining a state of well-differentiation. We asked if loss of NKX2-1 might also contribute to the release of ERK activity that drives tumor progression. Using human tissue samples and cell lines, xenografts, and genetic mouse models, we show that NKX2-1 induces the ERK phosphatase DUSP6. In cells with silenced NKX2-1, re-introduction of NKX2-1 induces DUSP6 and inhibits cell proliferation and migration and tumor growth and metastasis. Our CRISPR knockout studies show that DUSP6 is necessary for NKX2-1-mediated inhibition of ERK activity and tumor progression. Further, DUSP6 expression is sufficient to inhibit RAS-driven LUAD. We conclude that NKX2-1 silencing, and thereby DUSP6 silencing, is a mechanism by which early LUAD can unleash ERK hyperactivation for tumor progression.