The SOS1 Inhibitor MRTX0902 Blocks KRAS Activation and Demonstrates Antitumor Activity in Cancers Dependent on KRAS Nucleotide Loading.

KRAS is the most frequently mutated oncogene in human cancer and facilitates uncontrolled growth through hyperactivation of the receptor tyrosine kinase (RTK)/mitogen-activated protein kinase (MAPK) pathway. The Son of Sevenless homolog 1 (SOS1) protein functions as a guanine nucleotide exchange factor (GEF) for the RAS subfamily of small GTPases and represents a druggable target in the pathway. Using a structure-based drug discovery approach, MRTX0902 was identified as a selective and potent SOS1 inhibitor that disrupts the KRAS:SOS1 protein-protein interaction to prevent SOS1-mediated nucleotide exchange on KRAS and translates into an anti-proliferative effect in cancer cell lines with genetic alterations of the KRAS-MAPK pathway. MRTX0902 augmented the antitumor activity of the KRAS G12C inhibitor adagrasib when dosed in combination in eight out of 12 KRAS G12C-mutant human non-small cell lung cancer and colorectal cancer xenograft models. Pharmacogenomic profiling in preclinical models identified cell cycle genes and the SOS2 homolog as genetic co-dependencies and implicated tumor suppressor genes (NF1 and PTEN) in resistance following combination treatment. Lastly, combined vertical inhibition of RTK/MAPK pathway signaling by MRTX0902 with inhibitors of EGFR or RAF/MEK led to greater downregulation of pathway signaling and improved antitumor responses in KRAS-MAPK pathway-mutant models. These studies demonstrate the potential clinical application of dual inhibition of SOS1 and KRAS G12C and additional SOS1 combination strategies that will aide in the understanding of SOS1 and RTK/MAPK biology in targeted cancer therapy.

Preclinical in vitro and in vivo activity of the RAF/MEK clamp avutometinib in combination with FAK inhibition in uterine carcinosarcomas.

OBJECTIVES: Uterine carcinosarcomas (UCS) are rare, biologically aggressive tumors. Since UCS may harbor mutations in RAS/MAPK pathway genes we evaluated the preclinical in vitro and in vivo efficacy of the RAF/MEK clamp avutometinib in combination with the focal adhesion kinase (FAK) inhibitors defactinib or VS-4718 against multiple primary UCS cell lines and xenografts. METHODS: Whole-exome-sequencing (WES) was used to evaluate the genetic landscape of 5 primary UCS cell lines. The in vitro activity of avutometinib ± FAK inhibitor was evaluated using cell viability and cell cycle assays against primary UCS cell lines. Mechanistic studies were performed using western blot assays while in vivo experiments were completed in UCS tumor bearing mice treated with avutometinib ± FAK inhibitor by oral gavage. RESULTS: WES results demonstrated multiple UCS cell lines harbor genetic alterations including KRAS, PTK2, BRAF, MAP2K, and MAP2K1, potentially sensitizing to FAK and RAF/MEK inhibition. Four out of five of the UCS cell lines demonstrated in vitro sensitivity to FAK and/or RAF/MEK inhibition when used alone or in combination. By western blot assays, exposure of UCS cell lines to the combination of defactinib/avutometinib demonstrated decreased phosphorylated (p)-FAK as well as decreased p-ERK. In vivo, the combination of avutometinib/VS-4718 demonstrated superior tumor growth inhibition and longer survival compared to single agent treatment and controls starting at day 10 (p < 0.002) in UCS xenografts. CONCLUSION: The combination of avutometinib and defactinib demonstrates promising in vitro and in vivo anti-tumor activity against primary UCS cell lines and xenografts.

Preclinical efficacy of RAF/MEK clamp avutometinib in combination with FAK inhibition in low grade serous ovarian cancer.

OBJECTIVES: Low-grade-serous-ovarian-carcinoma (LGSOC) is characterized by a high recurrence rate and limited therapeutic options. About one-third of LGSOC contains mutations in MAPK pathway genes such as KRAS/NRAS/BRAF. Avutometinib is a dual RAF/MEK inhibitor while defactinib and VS-4718 are focal-adhesion-kinase-inhibitors (FAKi). We determined the preclinical efficacy of avutometinib±VS-4718 in LGSOC patient-derived-tumor-xenografts (PDX). METHODS: Whole-exome-sequencing (WES) was used to evaluate the genetic fingerprint of 3 patient-derived LGSOC (OVA(K)250, PERIT(M)17 and A(PE)148). OVA(K)250 tissue was successfully xenografted as PDX into female CB17/lcrHsd-Prkdc/SCID-mice. Animals were treated with either control, avutometinib, VS-4718, or avutometinib/ VS-4718 once daily five days on and two days off through oral gavage. Mechanistic studies were performed ex vivo using avutometinib±defactinib treated LGSOC tumor samples by western blot. RESULTS: WES results demonstrated wild-type KRAS in all 3 LGSOC. OVA(K)250 PDX showed gain-of-function mutations (GOF) in PTK2 and PTK2B genes, and loss-of-heterozygosity in ADRB2, potentially sensitizing to FAK and RAF/MEK inhibition. The combination of avutometinib/ VS-4718 demonstrated strong tumor-growth inhibition compared to controls starting at day 9 (p < 0.002) in OVA(K)250PDX. By 60 days, mice treated with avutometinib alone and avutometinib/VS-4718 were still alive; compared to median survival of 20 days in control-treated mice and of 35 days in VS-4718-treated mice (p < 0.0001). By western-blot assays exposure of OVA(K)250 to avutometinib, FAKi defactinib and their combination demonstrated decreased phosphorylated FAK (p-FAK) as well as decreased p-ERK. CONCLUSION: Avutometinib, and to a larger extent its combination with FAK inhibitor VS-4718, demonstrated promising in vivo activity against a KRAS wild-type LGSOC-PDX. These data support the ongoing registration-directed study (RAMP201/NCT04625270).

Duvelisib for Critically Ill Patients With Coronavirus Disease 2019: An Investigator-Initiated, Randomized, Placebo-Controlled, Double-Blind Pilot Trial.

Background: Despite improvements in prevention and treatment, severe coronavirus disease 2019 (COVID-19) is associated with high mortality. Phosphoinositide 3-kinase (PI3K) pathways contribute to cytokine and cell-mediated lung inflammation. We conducted a randomized, placebo-controlled, double-blind pilot trial to determine the feasibility, safety, and preliminary activity of duvelisib, a PI3Kδγ inhibitor, for the treatment of COVID-19 critical illness. Methods: We enrolled adults aged ≥18 years with a primary diagnosis of COVID-19 with hypoxic respiratory failure, shock, and/or new cardiac disease, without improvement after at least 48 hours of corticosteroid. Participants received duvelisib (25 mg) or placebo for up to 10 days. Participants had daily semi-quantitative viral load measurements performed. Dose modifications were protocol driven due to adverse events (AEs) or logarithmic change in viral load. The primary endpoint was 28-day overall survival (OS). Secondary endpoints included hospital and intensive care unit length of stay, 60-day OS, and duration of critical care interventions. Safety endpoints included viral kinetics and AEs. Exploratory endpoints included serial cytokine measurements and cytometric analysis. Results: Fifteen patients were treated in the duvelisib cohort, and 13 in the placebo cohort. OS at 28 days was 67% (95% confidence interval [CI], 38%-88%) compared to 62% (95% CI, 32%-86%) for placebo (P = .544). Sixty-day OS was 60% versus 46%, respectively (hazard ratio, 0.66 [95% CI, .22-1.96]; P = .454). Other secondary outcomes were comparable. Duvelisib was associated with lower inflammatory cytokines. Conclusions: In this pilot study, duvelisib did not significantly improve 28-day OS compared to placebo for severe COVID-19. Duvelisib appeared safe in this critically ill population and was associated with reduction in cytokines implicated in COVID-19 and acute respiratory distress syndrome, supporting further investigation. Clinical Trials Registration: NCT04372602.

High-throughput chemogenetic drug screening reveals PKC-RhoA/PKN as a targetable signaling vulnerability in GNAQ-driven uveal melanoma.

Uveal melanoma (UM) is the most prevalent cancer of the eye in adults, driven by activating mutation of GNAQ/GNA11; however, there are limited therapies against UM and metastatic UM (mUM). Here, we perform a high-throughput chemogenetic drug screen in GNAQ-mutant UM contrasted with BRAF-mutant cutaneous melanoma, defining the druggable landscape of these distinct melanoma subtypes. Across all compounds, darovasertib demonstrates the highest preferential activity against UM. Our investigation reveals that darovasertib potently inhibits PKC as well as PKN/PRK, an AGC kinase family that is part of the "dark kinome." We find that downstream of the Gαq-RhoA signaling axis, PKN converges with ROCK to control FAK, a mediator of non-canonical Gαq-driven signaling. Strikingly, darovasertib synergizes with FAK inhibitors to halt UM growth and promote cytotoxic cell death in vitro and in preclinical metastatic mouse models, thus exposing a signaling vulnerability that can be exploited as a multimodal precision therapy against mUM.

In vivo activity of the dual PI3Kδ and PI3Kγ inhibitor duvelisib against pediatric acute lymphoblastic leukemia xenografts.

BACKGROUND: Acute lymphoblastic leukemia (ALL) remains one of the most common causes of cancer-related mortality in children. Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases, and aberrations in the PI3K pathway are associated with several hematological malignancies, including ALL. Duvelisib (Copiktra) is an orally available, small molecule dual inhibitor of PI3Kδ and PI3Kγ, that is Food and Drug Administration (FDA) approved for the treatment of relapsed/refractory chronic lymphocytic leukemia and small lymphocytic lymphoma. Here, we report the efficacy of duvelisib against a panel of pediatric ALL patient-derived xenografts (PDXs). PROCEDURES: Thirty PDXs were selected for a single mouse trial based on PI3Kδ (PIK3CD) and PI3Kγ (PIK3CG) expression and mutational status. PDXs were grown orthotopically in NSG (NOD.Cg-Prkdcscid IL2rgtm1Wjl /SzJAusb) mice, and engraftment was evaluated by enumerating the proportion of human versus mouse CD45+ cells (%huCD45+ ) in the peripheral blood. Treatment commenced when the %huCD45+ reached greater than or equal to 1%, and events were predefined as %huCD45+ greater than or equal to 25% or leukemia-related morbidity. Duvelisib was administered per oral (50 mg/kg, twice daily for 28 days). Drug efficacy was assessed by event-free survival and stringent objective response measures. RESULTS: PI3Kδ and PI3Kγ mRNA expression was significantly higher in B-lineage than T-lineage ALL PDXs (p-values <.0001). Duvelisib was well-tolerated and reduced leukemia cells in the peripheral blood in four PDXs, but with only one objective response. There was no obvious relationship between duvelisib efficacy and PI3Kδ or PI3Kγ expression or mutation status, nor was the in vivo response to duvelisib subtype dependent. CONCLUSIONS: Duvelisib demonstrated limited in vivo activity against ALL PDXs.

Early path dominance as a principle for neurodevelopment.

We perform targeted attack, a systematic computational unlinking of the network, to analyze its effects on global communication across the brain network through its giant cluster. Across diffusion magnetic resonance images from individuals in the UK Biobank, Adolescent Brain Cognitive Development Study and Developing Human Connectome Project, we find that targeted attack procedures on increasing white matter tract lengths and densities are remarkably invariant to aging and disease. Time-reversing the attack computation suggests a mechanism for how brains develop, for which we derive an analytical equation using percolation theory. Based on a close match between theory and experiment, our results demonstrate that tracts are limited to emanate from regions already in the giant cluster and tracts that appear earliest in neurodevelopment are those that become the longest and densest.

Duvelisib Eliminates CLL B Cells, Impairs CLL-Supporting Cells, and Overcomes Ibrutinib Resistance in a Xenograft Model.

PURPOSE: Inhibitors of Bruton's tyrosine kinase (BTKi) and PI3K (PI3Ki) have significantly improved therapy of chronic lymphocytic leukemia (CLL). However, the emergence of resistance to BTKi has introduced an unmet therapeutic need. Hence, we sought evidence for essential roles of PI3K-δi and PI3K-γi in treatment-naïve and BTKi-refractory CLL. EXPERIMENTAL DESIGN: Responses to PI3K-δi, PI3K-γi, and the dual-inhibitor duvelisib in each B, T, and myeloid cell compartments of CLL were studied in vitro, and in a xenograft mouse model using primary cells from treatment-naïve and ibrutinib-resistant patients, and finally, in a patient with ibrutinib-resistant CLL treated with duvelisib. RESULTS: We demonstrate the essential roles of PI3K-δ for CLL B-cell survival and migration, of PI3K-γ for T-cell migration and macrophage polarization, and of dual inhibition of PI3K-δ,γ for efficacious reduction of leukemia burden. We also show that samples from patients whose disease progressed on ibrutinib were responsive to duvelisib therapy in a xenograft model, irrespective of BTK mutations. In support of this, we report a patient with ibrutinib-resistant CLL, bearing a clone with BTK and PLCγ2 mutations, who responded immediately to single-agent duvelisib with redistribution lymphocytosis followed by a partial clinical remission associated with modulation of T and myeloid cells. CONCLUSIONS: Our data define the mechanism of action whereby dual inhibition of PI3K-δ,γ affects CLL B-cell numbers and T and myeloid cell pro-leukemia functions and support the use of duvelisib as a valuable approach for therapeutic interventions, including for patients refractory to BTKi.

Roles of PI3Kγ and PI3Kδ in mantle cell lymphoma proliferation and migration contributing to efficacy of the PI3Kγ/δ inhibitor duvelisib.

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma that is incurable with existing therapies, and therefore presents a significant unmet clinical need. The ability of this disease to overcome therapy, including those that target the B cell receptor pathway which has a pathogenic role in MCL, highlights the need to develop new treatment strategies. Herein, we demonstrate that a distinguishing feature of lymph node resident MCL cells is the expression of phosphatidylinositol 3-kinase γ (PI3Kγ), a PI3K isoform that is not highly expressed in other B cells or B-cell malignancies. By exploring the role of PI3K in MCL using different PI3K isoform inhibitors, we provide evidence that duvelisib, a dual PI3Kδ/γ inhibitor, has a greater effect than PI3Kδ- and PI3Kγ-selective inhibitors in blocking the proliferation of primary MCL cells and MCL cell lines, and in inhibiting tumour growth in a mouse xenograft model. In addition, we demonstrated that PI3Kδ/γ signalling is critical for migration of primary MCL cells and cell lines. Our data indicates that aberrant expression of PI3Kγ is a critical feature of MCL pathogenesis. Thus, we suggest that the dual PI3Kδ/γ duvelisib would be effective for the treatment of mantle cell lymphoma.

Nonequilibrium statistical physics beyond the ideal heat bath approximation.

Important models of nonequilibrium statistical physics (NESP) are limited by a commonly used, but often unrecognized, near-equilibrium approximation. Fokker-Planck and Langevin equations, the Einstein and random-flight diffusion models, and the Schnakenberg model of biochemical networks suppose that fluctuations are due to an ideal equilibrium bath. But far from equilibrium, this perfect bath concept does not hold. A more principled approach should derive the rate fluctuations from an underlying dynamical model, rather than assuming a particular form. Here, using maximum caliber as the underlying principle, we derive corrections for NESP processes in an imperfect-but more realistic-environment, corrections which become particularly important for a system driven strongly away from equilibrium. Beyond characterizing a heat bath by the single equilibrium property of its temperature, the bath's speed and size must also be used to characterize the bath's ability to handle fast or large fluctuations.

Review of the application of Kirchhoff's Laws of series and parallel flows to pharmacology: Defining organ clearance.

Dosing rate decisions for drugs and changes in dosing in a patient due to disease states, drug interactions and pharmacogenomics are all based on clearance, a measure of the body's ability to eliminate drug. The primary organs of elimination are the liver and the kidney. Clearance for each of these organs is a summative composition of biologic processes. In 1857, Gustav Kirchhoff first developed his laws to describe the "motion of electricity in conductors... [and] ...in wires", recognizing that summative processes occur either in parallel or in series. Since then, Kirchhoff's Laws have also been applied to heat transfer, diffusion and drag force on falling objects, but not to pharmacology. Although not previously recognized, renal clearance always follow Kirchhoff's Laws, as does hepatic clearance for drugs where basolateral transporters are not clinically relevant. However, when basolateral transporters are clinically relevant, we demonstrate that the present accepted approach is inconsistent with recognized drug disposition processes. However, this clearance relationship can be easily corrected using Kirchhoff's Laws. The purpose of this review is to demonstrate that Kirchhoff's Laws, which define how to approach rate processes that occur in parallel versus processes that occur in series, can be applicable to pharmacology in addition to the over 160-year recognition of their use in physical sciences. We anticipate that the application to clearance will be only the first of many such pharmacological analyses.

Dual Stromal Targeting Sensitizes Pancreatic Adenocarcinoma for Anti-Programmed Cell Death Protein 1 Therapy.

BACKGROUND & AIMS: The stroma in pancreatic ductal adenocarcinoma (PDAC) contributes to its immunosuppressive nature and therapeutic resistance. Herein we sought to modify signaling and enhance immunotherapy efficacy by targeting multiple stromal components through both intracellular and extracellular mechanisms. METHODS: A murine liver metastasis syngeneic model of PDAC was treated with focal adhesion kinase inhibitor (FAKi), anti-programmed cell death protein 1 (PD-1) antibody, and stromal hyaluronan (HA) degradation by PEGylated recombinant human hyaluronidase (PEGPH20) to assess immune and stromal modulating effects of these agents and their combinations. RESULTS: The results showed that HA degradation by PEGPH20 and reduction in phosphorylated FAK expression by FAKi leads to improved survival in PDAC-bearing mice treated with anti-PD-1 antibody. HA degradation in combination with FAKi and anti-PD-1 antibody increases T-cell infiltration and alters T-cell phenotype toward effector memory T cells. FAKi alters the expression of T-cell modulating cytokines and leads to changes in T-cell metabolism and increases in effector T-cell signatures. HA degradation in combination with anti-PD-1 antibody and FAKi treatments reduces granulocytes, including granulocytic- myeloid-derived suppressor cells and decreases C-X-C chemokine receptor type 4 (CXCR4)-expressing myeloid cells, particularly the CXCR4-expressing granulocytes. Anti-CXCR4 antibody combined with FAKi and anti-PD-1 antibody significantly decreases metastatic rates in the PDAC liver metastasis model. CONCLUSIONS: This represents the first preclinical study to identify synergistic effects of targeting both intracellular and extracellular components within the PDAC stroma and supports testing anti-CXCR4 antibody in combination with FAKi as a PDAC treatment strategy.

Tumor FAK orchestrates immunosuppression in ovarian cancer via the CD155/TIGIT axis.

High-grade serous ovarian cancer (HGSOC) is a lethal malignancy characterized by an immunosuppressive tumor microenvironment containing few tumor infiltrating lymphocytes (TILs) and an insensitivity to checkpoint inhibitor immunotherapies. Gains in the PTK2 gene encoding focal adhesion kinase (FAK) at Chr8 q24.3 occur in ∼70% of HGSOC tumors, and elevated FAK messenger RNA (mRNA) levels are associated with poor patient survival. Herein, we show that active FAK, phosphorylated at tyrosine-576 within catalytic domain, is significantly increased in late-stage HGSOC tumors. Active FAK costained with CD155, a checkpoint receptor ligand for TIGIT (T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains), in HGSOC tumors and a selective association between FAK and TIGIT checkpoint ligands were supported by patient transcriptomic database analysis. HGSOC tumors with high FAK expression were associated with low CD3 mRNA levels. Accordingly, late-stage tumors showed elevated active FAK staining and significantly lower levels of CD3+ TILs. Using the KMF (Kras, Myc, FAK) syngeneic ovarian tumor model containing spontaneous PTK2 (FAK) gene gains, the effects of tumor intrinsic genetic or oral small molecule FAK inhibitior (FAKi; VS-4718) were evaluated in vivo. Blocking FAK activity decreased tumor burden, suppressed ascites KMF-associated CD155 levels, and increased peritoneal TILs. The combination of FAKi with blocking TIGIT antibody (1B4) maintained elevated TIL levels and reduced TIGIT+ T regulatory cell levels, prolonged host survival, increased CXCL13 levels, and led to the formation of omental tertiary lymphoid structures. Collectively, our studies support FAK and TIGIT targeting as a rationale immunotherapy combination for HGSOC.

PI3Kδ/γ inhibition promotes human CART cell epigenetic and metabolic reprogramming to enhance antitumor cytotoxicity.

Current limitations in using chimeric antigen receptor T(CART) cells to treat patients with hematological cancers include limited expansion and persistence in vivo that contribute to cancer relapse. Patients with chronic lymphocytic leukemia (CLL) have terminally differentiated T cells with an exhausted phenotype and experience low complete response rates after autologous CART therapy. Because PI3K inhibitor therapy is associated with the development of T-cell-mediated autoimmunity, we studied the effects of inhibiting the PI3Kδ and PI3Kγ isoforms during the manufacture of CART cells prepared from patients with CLL. Dual PI3Kδ/γ inhibition normalized CD4/CD8 ratios and maximized the number of CD8+ T-stem cell memory, naive, and central memory T-cells with dose-dependent decreases in expression of the TIM-3 exhaustion marker. CART cells manufactured with duvelisib (Duv-CART cells) showed significantly increased in vitro cytotoxicity against CD19+ CLL targets caused by increased frequencies of CD8+ CART cells. Duv-CART cells had increased expression of the mitochondrial fusion protein MFN2, with an associated increase in the relative content of mitochondria. Duv-CART cells exhibited increased SIRT1 and TCF1/7 expression, which correlated with epigenetic reprograming of Duv-CART cells toward stem-like properties. After transfer to NOG mice engrafted with a human CLL cell line, Duv-CART cells expressing either a CD28 or 41BB costimulatory domain demonstrated significantly increased in vivo expansion of CD8+ CART cells, faster elimination of CLL, and longer persistence. Duv-CART cells significantly enhanced survival of CLL-bearing mice compared with conventionally manufactured CART cells. In summary, exposure of CART to a PI3Kδ/γ inhibitor during manufacturing enriched the CART product for CD8+ CART cells with stem-like qualities and enhanced efficacy in eliminating CLL in vivo.

FAK inhibition alone or in combination with adjuvant therapies reduces cancer stem cell activity.

Cancer stem-like cells (CSC) contribute to therapy resistance and recurrence. Focal adhesion kinase (FAK) has a role in CSC regulation. We determined the effect of FAK inhibition on breast CSC activity alone and in combination with adjuvant therapies. FAK inhibition reduced CSC activity and self-renewal across all molecular subtypes in primary human breast cancer samples. Combined FAK and paclitaxel reduced self-renewal in triple negative cell lines. An invasive breast cancer cohort confirmed high FAK expression correlated with increased risk of recurrence and reduced survival. Co-expression of FAK and CSC markers was associated with the poorest prognosis, identifying a high-risk patient population. Combined FAK and paclitaxel treatment reduced tumour size, Ki67, ex-vivo mammospheres and ALDH+ expression in two triple negative patient derived Xenograft (PDX) models. Combined treatment reduced tumour initiation in a limiting dilution re-implantation PDX model. Combined FAK inhibition with adjuvant therapy has the potential to improve breast cancer survival.

Synergistic efficacy of the dual PI3K-δ/γ inhibitor duvelisib with the Bcl-2 inhibitor venetoclax in Richter syndrome PDX models.

A small subset of cases of chronic lymphocytic leukemia undergoes transformation to diffuse large B-cell lymphoma, Richter syndrome (RS), which is associated with a poor prognosis. Conventional chemotherapy results in limited responses, underlining the need for novel therapeutic strategies. Here, we investigate the ex vivo and in vivo efficacy of the dual phosphatidylinositol 3-kinase-δ/γ (PI3K-δ/γ) inhibitor duvelisib (Duv) and the Bcl-2 inhibitor venetoclax (Ven) using 4 different RS patient-derived xenograft (PDX) models. Ex vivo exposure of RS cells to Duv, Ven, or their combination results in variable apoptotic responses, in line with the expression levels of target proteins. Although RS1316, IP867/17, and RS9737 cells express PI3K-δ, PI3K-γ, and Bcl-2 and respond to the drugs, RS1050 cells, expressing very low levels of PI3K-γ and lacking Bcl-2, are fully resistant. Moreover, the combination of these drugs is more effective than each agent alone. When tested in vivo, RS1316 and IP867/17 show the best tumor growth inhibition responses, with the Duv/Ven combination leading to complete remission at the end of treatment. The synergistic effect of Duv and Ven relies on the crosstalk between PI3K and apoptotic pathways occurring at the GSK3ß level. Indeed, inhibition of PI3K signaling by Duv results in GSK3ß activation, leading to ubiquitination and subsequent degradation of both c-Myc and Mcl-1, making RS cells more sensitive to Bcl-2 inhibition by Ven. This work provides, for the first time, a proof of concept of the efficacy of dual targeting of PI3K-δ/γ and Bcl-2 in RS and providing an opening for a Duv/Ven combination for these patients. Clinical studies in aggressive lymphomas, including RS, are under way. This trial was registered at www.clinicaltrials.gov as #NCT03892044.

Synthetic Lethal Screens Reveal Cotargeting FAK and MEK as a Multimodal Precision Therapy for GNAQ-Driven Uveal Melanoma.

PURPOSE: Uveal melanoma is the most common eye cancer in adults. Approximately 50% of patients with uveal melanoma develop metastatic uveal melanoma (mUM) in the liver, even after successful treatment of the primary lesions. mUM is refractory to current chemo- and immune-therapies, and most mUM patients die within a year. Uveal melanoma is characterized by gain-of-function mutations in GNAQ/GNA11, encoding Gαq proteins. We have recently shown that the Gαq-oncogenic signaling circuitry involves a noncanonical pathway distinct from the classical activation of PLCß and MEK-ERK. GNAQ promotes the activation of YAP1, a key oncogenic driver, through focal adhesion kinase (FAK), thereby identifying FAK as a druggable signaling hub downstream from GNAQ. However, targeted therapies often activate compensatory resistance mechanisms leading to cancer relapse and treatment failure. EXPERIMENTAL DESIGN: We performed a kinome-wide CRISPR-Cas9 sgRNA screen to identify synthetic lethal gene interactions that can be exploited therapeutically. Candidate adaptive resistance mechanisms were investigated by cotargeting strategies in uveal melanoma and mUM in vitro and in vivo experimental systems. RESULTS: sgRNAs targeting the PKC and MEK-ERK signaling pathways were significantly depleted after FAK inhibition, with ERK activation representing a predominant resistance mechanism. Pharmacologic inhibition of MEK and FAK showed remarkable synergistic growth-inhibitory effects in uveal melanoma cells and exerted cytotoxic effects, leading to tumor collapse in uveal melanoma xenograft and liver mUM models in vivo. CONCLUSIONS: Coupling the unique genetic landscape of uveal melanoma with the power of unbiased genetic screens, our studies reveal that FAK and MEK-ERK cotargeting may provide a new network-based precision therapeutic strategy for mUM treatment.See related commentary by Harbour, p. 2967.

MEK inhibitors reduce cellular expression of ACE2, pERK, pRb while stimulating NK-mediated cytotoxicity and attenuating inflammatory cytokines relevant to SARS-CoV-2 infection.

COVID-19 affects vulnerable populations including elderly individuals and patients with cancer. Natural Killer (NK) cells and innate-immune TRAIL suppress transformed and virally-infected cells. ACE2, and TMPRSS2 protease promote SARS-CoV-2 infectivity, while inflammatory cytokines IL-6, or G-CSF worsen COVID-19 severity. We show MEK inhibitors (MEKi) VS-6766, trametinib and selumetinib reduce ACE2 expression in human cells. In some human cells, remdesivir increases ACE2-promoter luciferase-reporter expression, ACE2 mRNA and protein, and ACE2 expression is attenuated by MEKi. In serum-deprived and stimulated cells treated with remdesivir and MEKi we observed correlations between pRB, pERK, and ACE2 expression further supporting role of proliferative state and MAPK pathway in ACE2 regulation. We show elevated cytokines in COVID-19-(+) patient plasma (N = 9) versus control (N = 11). TMPRSS2, inflammatory cytokines G-CSF, M-CSF, IL-1α, IL-6 and MCP-1 are suppressed by MEKi alone or with remdesivir. We observed MEKi stimulation of NK-cell killing of target-cells, without suppressing TRAIL-mediated cytotoxicity. Pseudotyped SARS-CoV-2 virus with a lentiviral core and SARS-CoV-2 D614 or G614 SPIKE (S) protein on its envelope infected human bronchial epithelial cells, small airway epithelial cells, or lung cancer cells and MEKi suppressed infectivity of the pseudovirus. We show a drug class-effect with MEKi to stimulate NK cells, inhibit inflammatory cytokines and block host-factors for SARS-CoV-2 infection leading also to suppression of SARS-CoV-2-S pseudovirus infection of human cells. MEKi may attenuate SARS-CoV-2 infection to allow immune responses and antiviral agents to control disease progression.

Natural Killer cell activation, reduced ACE2, TMPRSS2, cytokines G-CSF, M-CSF and SARS-CoV-2-S pseudovirus infectivity by MEK inhibitor treatment of human cells.

COVID-19 affects vulnerable populations including elderly individuals and patients with cancer. Natural Killer (NK) cells and innate-immune TRAIL suppress transformed and virally-infected cells. ACE2, and TMPRSS2 protease promote SARS-CoV-2 infectivity, while inflammatory cytokines IL-6, or G-CSF worsen COVID-19 severity. We show MEK inhibitors (MEKi) VS-6766, trametinib and selumetinib reduce ACE2 expression in human cells. In some human cells, remdesivir increases ACE2-promoter luciferase-reporter expression, ACE2 mRNA and protein, and ACE2 expression is attenuated by MEKi. In serum-deprived and stimulated cells treated with remdesivir and MEKi we observed correlations between pRB, pERK, and ACE2 expression further supporting role of proliferative state and MAPK pathway in ACE2 regulation. We show elevated cytokines in COVID-19-(+) patient plasma (N=9) versus control (N=11). TMPRSS2, inflammatory cytokines G-CSF, M-CSF, IL-1α, IL-6 and MCP-1 are suppressed by MEKi alone or with remdesivir. We observed MEKi stimulation of NK-cell killing of target-cells, without suppressing TRAIL-mediated cytotoxicity. Pseudotyped SARS-CoV-2 virus with a lentiviral core and SARS-CoV-2 D614 or G614 SPIKE (S) protein on its envelope infected human bronchial epithelial cells, small airway epithelial cells, or lung cancer cells and MEKi suppressed infectivity of the pseudovirus. We show a drug class-effect with MEKi to stimulate NK cells, inhibit inflammatory cytokines and block host-factors for SARS-CoV-2 infection leading also to suppression of SARS-CoV-2-S pseudovirus infection of human cells. MEKi may attenuate SARS-CoV-2 infection to allow immune responses and antiviral agents to control disease progression.