Small-molecule Kinase Inhibitors Research Articles

Abstract 10701: Suspension Differentiation of Cardiomyocyte Spheroids Improves Quality and Yields

Introduction: Improvements in stem cell viability, differentiation efficiency, and functional activity opened the door for advanced research applications in cardiac regeneration. However, the use of cell-based cardiac therapies, including implantable patches and cell products as well as in-vitro models for mechanistic studies and drug development requires large numbers of high-quality cardiomyocytes (CMs). Typically, differentiation is conducted in 6-well culture plates via the GiWi method, which uses small-molecule inhibitors of glycogen synthase kinase (GSK) and tankyrase to first activate and then suppress Wnt signaling. This monolayer method requires significant manual labor and cannot be easily scaled. Suspension culture and differentiation of free-floating hiPSC aggregates following similar biochemical cues offers advantages for commercial biomanufacturing purposes and provides a more physiological environment for cell development. Hypothesis: We assessed the hypothesis that when hiPSCs are cultured and differentiated in suspension they will produce CMs with improved yield, purity, and quality. Methods: hiPSCs were expanded as aggregates in culture flask using a fed-batch approach then differentiated over a 12 day period with Wnt activation on day 0 and inhibition on day 3. Optimization studies examined purity on day 9 and complete characterization via morphological assessments and CM-specific genes expression was done on day 12 following metabolic purification. All studies were conducted with a minimum of 4 replicates. Results: Optimal conditions for both purity and yield based on cardiac troponin T staining were achieved with an initial cell density of 1.6x10 6 cells/mL, 6 μM CHIR99021, and 55 rpm shaking. After scaling to 30mL culture volume the purity of hiPSC-CMs differentiated via our novel protocol was 98.2±0.8% with yields of 1.47±0.18 million cells/mL and less between-batch purity variability than hiPSC-CMs produced in 2D cultures. Conclusions: Suspension differentiation of hiPSCs into CMs in a widely available format results in improved biomanufacturing endpoints. Therefore, this method functions as a groundwork for future bioreactor systems to produce the large number of cells needed for clinical applications.

Targeted Therapies in Cancer: To Be or Not to Be, Selective.

Development of targeted therapies in recent years revealed several nonchemotherapeutic options for patients. Chief among targeted therapies is small molecule kinase inhibitors targeting key oncogenic signaling proteins. Through competitive and noncompetitive inhibition of these kinases, and therefore the pathways they activate, cancers can be slowed or completely eradicated, leading to partial or complete remissions for many cancer types. Unfortunately, for many patients, resistance to targeted therapies, such as kinase inhibitors, ultimately develops and can necessitate multiple lines of treatment. Drug resistance can either be de novo or acquired after months or years of drug exposure. Since resistance can be due to several unique mechanisms, there is no one-size-fits-all solution to this problem. However, combinations that target complimentary pathways or potential escape mechanisms appear to be more effective than sequential therapy. Combinations of single kinase inhibitors or alternately multikinase inhibitor drugs could be used to achieve this goal. Understanding how to efficiently target cancer cells and overcome resistance to prior lines of therapy became imperative to the success of cancer treatment. Due to the complexity of cancer, effective treatment options in the future will likely require mixing and matching these approaches in different cancer types and different disease stages.

Discovery of a novel potentially transforming somatic mutation in CSF2RB gene in breast cancer.

The colony stimulating factor 2 receptor subunit beta (CSF2RB) is the common signaling subunit of the cytokine receptors for IL‐3, IL‐5, and GM‐CSF. Several studies have shown that spontaneous and random mutants of CSF2RB can lead to ligand independence in vitro. To date, no report(s) have been shown for the presence of potentially transforming and oncogenic CSF2RB mutation(s) clinically in cancer patients until the first reported case of a leukemia patient in 2016 harboring a germline‐activating mutation (R461C). We combined exome sequencing, pathway analyses, and functional assays to identify novel somatic mutations in KAIMRC1 cells and breast tumor specimen. The patient’s peripheral blood mononuclear cell (PBMC) exome served as a germline control in the identification of somatic mutations. Here, we report the discovery of a novel potentially transforming and oncogenic somatic mutation (S230I) in the CSF2RB gene of a breast cancer patient and the cell line, KAIMRC1 established from her breast tumor tissue. KAIMRC1 cells are immortalized and shown to survive and proliferate in ligand starvation condition. Immunoblot analysis showed that mutant CSF2RB signals through JAK2/STAT and PI3K/mTOR pathways in ligand starvation conditions. Screening a small molecule kinase inhibitor library revealed potent JAK2 inhibitors against KAIMRC1 cells. We, for the first time, identified a somatic, potentially transforming, and oncogenic CSF2RB mutation (S230I) in breast cancer patients that seem to be an actionable mutation leading to the development of new therapeutics for breast cancer.

Small Molecule Kinase Inhibitor Drugs (1995-2021): Medical Indication, Pharmacology, and Synthesis.

The central role of dysregulated kinase activity in the etiology of progressive disorders, including cancer, has fostered incremental efforts on drug discovery programs over the past 40 years. As a result, kinase inhibitors are today one of the most important classes of drugs. The FDA approved 73 small molecule kinase inhibitor drugs until September 2021, and additional inhibitors were approved by other regulatory agencies during that time. To complement the published literature on clinical kinase inhibitors, we have prepared a review that recaps this large data set into an accessible format for the medicinal chemistry community. Along with the therapeutic and pharmacological properties of each kinase inhibitor approved across the world until 2020, we provide the synthesis routes originally used during the discovery phase, many of which were only available in patent applications. In the last section, we also provide an update on kinase inhibitor drugs approved in 2021.

Evaluation of small molecule kinase inhibitors as novel antimicrobial and antibiofilm agents.

Antibiotic resistance is a global and pressing concern. Our current therapeutic arsenal is increasingly limited as bacteria are developing resistance at a rate that far outpaces our ability to create new treatments. Novel approaches to treating and curing bacterial infections are urgently needed. Bacterial kinases have been increasingly explored as novel drug targets and are poised for development into novel therapeutic agents to combat bacterial infections. This review describes several general classes of bacterial kinases that play important roles in bacterial growth, antibiotic resistance, and biofilm formation. General features of these kinase classes are discussed and areas of particular interest for the development of inhibitors will be highlighted. Small molecule kinase inhibitors are described and organized by phenotypic effect, spotlighting particularly interesting inhibitors with novel functions and potential therapeutic benefit. Finally, we provide our perspective on the future of bacterial kinase inhibition as a viable strategy to combat bacterial infections and overcome the pressures of increasing antibiotic resistance.

A RARE CAUSE OF INTERSTITIAL LUNG DISEASE

TOPIC: Diffuse Lung Disease TYPE: Medical Student/Resident Case Reports INTRODUCTION: Clinicians should be aware of the potential adverse effects and complications that can occur with molecularly targeted agents for them to be able to diagnose and treat the condition. CASE PRESENTATION: 65-year-old female with stage IV epidermal growth factor receptor (EGFR) lung adenocarcinoma with metastases to the brain, spine, liver, and kidneys undergoing treatment with osimertinib for two months presented with a subjective fever of 102 °F. On admission the patient was tachycardic with an elevated lactate warranting investigation and treatment of sepsis secondary to a UTI. Despite using broad spectrum antibiotics and proper fluid resuscitation, her hospital course was complicated with the development of a new onset dyspnea with exertion and insidious hypoxic respiratory failure. Computed tomography angiography (CTA) of the chest revealed an incidental sub segmental pulmonary embolism which was not treated with anticoagulation due to underlying hemorrhagic brain metastasis. More importantly, the CTA showed new ground glass opacities demonstrated on the upper lung fields concerning for interstitial lung disease (ILD). Osimertinib was discontinued and high-dose IV methylprednisolone was administered resulting in immediate relief of dyspnea and cessation of relapsing fever. There was also a significant improvement to the hypoxia within a 24-hour period. Patient was transitioned to oral prednisone and discharged home the following day. DISCUSSION: Anti-epidermal growth factor receptor agents, a class of small molecule kinase inhibitors, are primarily used for the treatment of advanced non-small cell lung cancer. Common side effects for this particular class of tyrosine kinase inhibitors (TKIs) include fatigue, rashes, and lymphopenia. The risk of ILD for patient's taking EGFR-TKIs is a rare incidence, occurring at a rate of 1-3%, within the first 2-3 months of initiating therapy. The exact mechanism of TKI induced lung injury remains unknown, the proposing theory is an interruption to the alveolar repair mechanism which potentiates lung injury to other causes, such as sepsis, medications, and prior radiation therapy. Discontinuation of EGFR-TKIs therapy is the primary modality to prevent worsening of ILD. Utilization of high-dose glucocorticoids is an additional measure that can be employed for the treatment of drug-induced ILD. CONCLUSIONS: Physicians must be vigilant, with a high degree of clinical suspicion, to discontinue the offending agent in the event that a patient on EGFR-TKIs therapy presents with fever, acute dyspnea with or without cough, tachycardia, and hypoxia. If symptoms of the adverse reaction are not recognized early enough, they may progress to other conditions, such as pulmonary fibrosis. In addition, there is an increased mortality ratewith the continuation of these agents once ILD develops. REFERENCE #1: Severe immune-related adverse events are common with sequential PD-(L)1 blockade and osimertinib.Ann Oncol. 2019;30(5):839. DISCLOSURES: No relevant relationships by Ankita Aggarwal, source=Web Response No relevant relationships by Yashar Eshman, source=Web Response no disclosure on file for Mohammad Fityan; No relevant relationships by Victoria Gonzalez, source=Web Response No relevant relationships by Zain Kulairi, source=Web Response

Regulation of Mitogen-Activated Protein Kinase Signaling Pathways by the Ubiquitin-Proteasome System and Its Pharmacological Potential.

Mitogen-activated protein kinase (MAPK) cascades are evolutionarily conserved signaling pathways that play essential roles in transducing extracellular environmental signals into diverse cellular responses to maintain homeostasis. These pathways are classically organized into an architecture of three sequentially acting protein kinases: a MAPK kinase kinase that phosphorylates and activates a MAPK kinase, which in turn phosphorylates and activates the effector MAPK. The activity of MAPKs is tightly regulated by phosphorylation of their activation loop, which can be modulated by positive and negative feedback mechanisms to control the amplitude and duration of the signal. The signaling outcomes of MAPK pathways are further regulated by interactions of MAPKs with scaffolding and regulatory proteins. Accumulating evidence indicates that, in addition to these mechanisms, MAPK signaling is commonly regulated by ubiquitin-proteasome system (UPS)-mediated control of the stability and abundance of MAPK pathway components. Notably, the biologic activity of some MAPKs appears to be regulated mainly at the level of protein turnover. Recent studies have started to explore the potential of targeted protein degradation as a powerful strategy to investigate the biologic functions of individual MAPK pathway components and as a new therapeutic approach to overcome resistance to current small-molecule kinase inhibitors. Here, we comprehensively review the mechanisms, physiologic importance, and pharmacological potential of UPS-mediated protein degradation in the control of MAPK signaling. SIGNIFICANCE STATEMENT: Accumulating evidence highlights the importance of targeted protein degradation by the ubiquitin-proteasome system in regulating and fine-tuning the signaling output of mitogen-activated protein kinase (MAPK) pathways. Manipulating protein levels of MAPK cascade components may provide a novel approach for the development of selective pharmacological tools and therapeutics.

Small molecule kinase inhibitors enhance aminolevulinic acid-mediated protoporphyrin IX fluorescence and PDT response in triple negative breast cancer cell lines.

.Significance: We demonstrate that clinically used kinase inhibitors such as lapatinib can be used for enhancing aminolevulinic acid (ALA) for tumor fluorescence imaging and photodynamic therapy (PDT).Aim: ALA is used as a prodrug for protoporphyrin IX (PpIX) fluorescence-guided tumor resection and PDT. Our previous studies indicate that tumors with high ABCG2 activity exhibit low PpIX fluorescence, which hampers the application of ALA. We aim to determine whether clinically used ABCG2-interacting kinase inhibitors increase ALA-PpIX fluorescence and PDT.Approach: PpIX fluorescence was determined by spectrofluorometry, flow cytometry, and confocal microscopy after ALA alone or in combination with kinase inhibitors in triple negative breast cancer (TNBC) cell lines. Cytotoxicity was examined after ALA-PDT alone or in combination with kinase inhibitors. Effect of single and combination treatments on apoptosis was assessed by Western blot.Results: Four kinase inhibitors (lapatinib, PD169316, sunitinib, gefitinib) significantly increased ALA-PpIX fluorescence and PDT response in TNBC cells with ABCG2 activity, but not in MCF10A nontumor breast epithelial cell line without ABCG2 activity. Confocal microscopic imaging showed that PpIX fluorescence was weak and diffuse after ALA alone, which was greatly enhanced by kinase inhibitors, particularly in the mitochondria. Lapatinib was the only inhibitor that significantly reduced PpIX efflux in cell culture medium and showed stronger enhancement of PDT response than other kinase inhibitors. Lapatinib, in combination with ALA, induced tumor cells to undergo apoptosis, whereas no apoptosis was detected after each individual treatment.Conclusions: Although all four kinase inhibitors were able to enhance ALA-PpIX fluorescence and PDT, lapatinib exhibited the strongest enhancement effect. As an FDA-approved kinase inhibitor for breast cancer treatment, lapatinib is ready to be used in combination with ALA for therapeutic enhancement in tumors with elevated ABCG2 activity. This rational combination approach warrants further investigation in tumor models.

MPN-161: A Phase I Study of TP-3654, an Oral PIM Kinase Inhibitor, in Patients with Intermediate-2 or High-Risk Primary or Secondary Myelofibrosis (MF)

Context: MF is characterized by bone marrow fibrosis (BMF), ineffective hematopoiesis, splenomegaly, and debilitating symptoms. Expression of PIM-1 is significantly upregulated in MF hematopoietic cells, indicating that PIM-1 is a potential therapeutic target in MF. TP-3654, an oral small-molecule investigational PIM-1 kinase inhibitor with favorable selectivity against other kinases, has been shown to inhibit proliferation and increase apoptosis in murine and human hematopoietic cells expressing the JAK2V617F mutation. TP-3654 alone and in combination with ruxolitinib normalizes leukocyte counts and reduces spleen size and BMF in JAK2V617F and MPLW515L murine MF models. Objective: To evaluate TP-3654 monotherapy in patients with MF. Design: Phase I, open-label, dose-escalation study (NCT04176198). Setting: Multicenter, US only. Patients or Other Participants: Key eligibility criteria include primary or secondary MF (post-polycythemia vera-MF/post-essential thrombocythemia-MF); intermediate-2/high-risk MF per Dynamic International Prognostic Scoring System (DIPSS); previously failed or are ineligible to receive Janus kinase inhibitors (e.g., ruxolitinib, fedratinib); grade ≥2 BMF; platelet count ≥50x109/L; absolute neutrophil count ≥1x109/L; peripheral blood blast counts Interventions: Patients receive oral TP-3654. Dose escalation, performed using a Bayesian Logistic Regression Model with Escalation with Overdose Control, will continue until identification of the maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D) is achieved. Dose expansion will follow. Main Outcomes Measures: Primary objectives: to determine MTD and/or identify the RP2D of TP-3654 monotherapy. Secondary objectives: to assess preliminary activity (spleen volume reduction, symptom improvement, and BMF reduction), determine QT interval changes, and establish the pharmacokinetic profile. Exploratory objectives: to study pharmacodynamic markers in peripheral blood and BM biopsies. This study is currently recruiting.

Acetylation of the Catalytic Lysine Inhibits Kinase Activity in PI3Kδ

Covalent inhibition is a powerful strategy to develop potent and selective small molecule kinase inhibitors. Targeting the conserved catalytic lysine is an attractive method for selective kinase inactivation. We have developed novel, selective inhibitors of phosphoinositide 3-kinase δ (PI3Kδ) which acylate the catalytic lysine, Lys779, using activated esters as the reactive electrophiles. The acylating agents were prepared by adding the activated ester motif to a known selective dihydroisobenzofuran PI3Kδ inhibitor. Three esters were designed, including an acetate ester which was the smallest lysine modification evaluated in this work. Covalent binding to the enzyme was characterized by intact protein mass spectrometry of the PI3Kδ-ester adducts. An enzymatic digest coupled with tandem mass spectrometry identified Lys779 as the covalent binding site, and a biochemical activity assay confirmed that PI3Kδ inhibition was a direct result of covalent lysine acylation. These results indicate that a simple chemical modification such as lysine acetylation is sufficient to inhibit kinase activity. The selectivity of the compounds was evaluated against lipid kinases in cell lysates using a chemoproteomic binding assay. Due to the conserved nature of the catalytic lysine across the kinome, we believe the covalent inhibition strategy presented here could be applicable to a broad range of clinically relevant targets.

Assessing the Inhibitory Potential of Kinase Inhibitors In Vitro: Major Pitfalls and Suggestions for Improving Comparability of Data Using CK1 Inhibitors as an Example.

Phosphorylation events catalyzed by protein kinases represent one of the most prevalent as well as important regulatory posttranslational modifications, and dysregulation of protein kinases is associated with the pathogenesis of different diseases. Therefore, interest in developing potent small molecule kinase inhibitors has increased enormously within the last two decades. A critical step in the development of new inhibitors is cell-free in vitro testing with the intention to determine comparable parameters like the commonly used IC50 value. However, values described in the literature are often biased as experimental setups used for determination of kinase activity lack comparability due to different readout parameters, insufficient normalization or the sheer number of experimental approaches. Here, we would like to hold a brief for highly sensitive, radioactive-based in vitro kinase assays especially suitable for kinases exhibiting autophosphorylation activity. Therefore, we demonstrate a systematic workflow for complementing and validating results from high-throughput screening as well as increasing the comparability of enzyme-specific inhibitor parameters for radiometric as well as non-radiometric assays. Using members of the CK1 family of serine/threonine-specific protein kinases and established CK1-specific inhibitors as examples, we clearly demonstrate the power of our proposed workflow, which has the potential to support the generation of more comparable data for biological characterization of kinase inhibitors.

Targeted activity of the small molecule kinase inhibitor Pz-1 towards RET and TRK kinases

We have recently described Pz-1, a benzimidazole-based type-2 RET and VEGFR2 inhibitor. Based on a kinome scan, here we show that Pz-1 is also a potent (IC50 < 1 nM) TRKA/B/C inhibitor. Pz-1 potently inhibited proliferation of human cancer cells carrying either RET- or TRKA oncoproteins (IC50 ~ 1 nM), with a negligible effect against RET- and TRKA-negative cells. By testing mutations, known to mediate resistance to other compounds, RET G810R/S, but not L730I/V, E732K, V738A and Y806N, showed some degree of resistance to Pz-1. In the case of TRKA, G595R and F589L, but not G667C, showed some degree of resistance. In xenograft models, orally administered Pz-1 almost completely inhibited RET- and TRKA-mutant tumours at 1–3 mg/kg/day but showed a reduced effect on RET/TRKA-negative cancer models. The activity, albeit reduced, on RET/TRKA-negative tumours may be justified by VEGFR2 inhibition. Tumours induced by NIH3T3 cells transfected by RET G810R and TRKA G595R featured resistance to Pz-1, demonstrating that RET or TRKA inhibition is critical for its anti-tumourigenic effect. In conclusion, Pz-1 represents a new powerful kinase inhibitor with distinct activity towards cancers induced by oncogenic RET and TRKA variants, including some mutants displaying resistance to other drugs.

Trends in kinase drug discovery: targets, indications and inhibitor design.

The FDA approval of imatinib in 2001 was a breakthrough in molecularly targeted cancer therapy and heralded the emergence of kinase inhibitors as a key drug class in the oncology area and beyond. Twenty years on, this article analyses the landscape of approved and investigational therapies that target kinases and trends within it, including the most popular targets of kinase inhibitors and their expanding range of indications. There are currently 71 small-molecule kinase inhibitors (SMKIs) approved by the FDA and an additional 16 SMKIs approved by other regulatory agencies. Although oncology is still the predominant area for their application, there have been important approvals for indications such as rheumatoid arthritis, and one-third of the SMKIs in clinical development address disorders beyond oncology. Information on clinical trials of SMKIs reveals that approximately 110 novel kinases are currently being explored as targets, which together with the approximately 45 targets of approved kinase inhibitors represent only about 30% of the human kinome, indicating that there are still substantial unexplored opportunities for this drug class. We also discuss trends in kinase inhibitor design, including the development of allosteric and covalent inhibitors, bifunctional inhibitors and chemical degraders.

A potential signaling axis between RON kinase receptor and hypoxia-inducible factor-1 alpha in pancreatic cancer.

The Cancer Genome Atlas (TCGA) of a pancreatic cancer cohort identified high MST1R (RON tyrosine kinase receptor) expression correlated with poor prognosis in human pancreatic cancer. RON expression is null/minimal in normal pancreas but elevates from pan‐in lesions through invasive carcinomas. We report using multiple approaches RON directly regulates HIF‐1α, a critical driver of genes involved in cancer cell invasion and metastasis. RON and HIF‐1α are highly co‐expressed in the 101 human PDAC tumors analyzed and RON expression correlated with HIF‐1α expression in a subset of PDAC cell lines. knockdown of RON expression in RON positive cells blocked HIF‐1α expression, whereas ectopic RON expression in RON null cells induced HIF‐1α expression suggesting the direct regulation of HIF‐1α by RON kinase receptor. RON regulates HIF‐1α through an unreported transcriptional mechanism involving PI3 kinase‐mediated AKT phosphorylation and Sp1‐dependent HIF‐1α promoter activity leading to increased HIF‐1α mRNA expression. RON/HIF‐1α modulation altered the invasive behavior of PDAC cells. A small‐molecule RON kinase inhibitor decreased RON ligand, MSP‐induced HIF‐1α expression, and invasion of PDAC cells. Immunohistochemical analysis on RON knockdown orthotopic PDAC tumor xenograft confirmed that RON inhibition significantly blocked HIF‐1α expression. RON/HIF‐1α co‐expression also exists in triple‐negative breast cancer cells, a tumor type that also lacks molecular therapeutic targets. This is the first report describing RON/HIF‐1α axis in any tumor type and is a potential novel therapeutic target.

Blocking Short-Form Ron Eliminates Breast Cancer Metastases through Accumulation of Stem-Like CD4+ T Cells That Subvert Immunosuppression.

The discovery that SF-Ron promotes antitumor immune responses has significant clinical implications. Therapeutic antibodies targeting full-length Ron may not be effective for immunotherapy; poor efficacy of such antibodies in trials may be due to their inability to block SF-Ron. Our data warrant trials with inhibitors targeting SF-Ron in combination with immunotherapy. This article is highlighted in the In This Issue feature, p. 2945.

Characterisation of an enhanced preclinical model of experimental MPO-ANCA autoimmune vasculitis.

Experimental autoimmune vasculitis (EAV) is a model of antineutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) induced by immunisation of susceptible rat strains with myeloperoxidase (MPO). Animals develop circulating MPO-ANCA, pulmonary haemorrhage, and glomerulonephritis, although renal injury is mild and recovers spontaneously without treatment. In this study we aimed to augment the severity of glomerulonephritis. Following induction of EAV on day 0, a sub-nephritogenic dose of nephrotoxic serum (NTS) containing heterologous antibodies to glomerular basement membrane was administered on day 14. This resulted in a significant increase in disease severity at day 28 compared to MPO immunisation alone - with more urinary abnormalities, infiltrating glomerular leucocytes, and crescent formation that progressed to glomerular and tubulointerstitial scarring by day 56, recapitulating important features of human disease. Importantly, the glomerulonephritis remained pauci-immune, and was strictly dependent on the presence of autoimmunity to MPO, as there was no evidence of renal disease following administration of sub-nephritogenic NTS alone or after immunisation with a control protein in place of MPO. Detailed phenotyping of glomerular leucocytes identified an early infiltrate of non-classical monocytes following NTS administration that, in the presence of autoimmunity to MPO, may initiate the subsequent influx of classical monocytes which augment glomerular injury. We also showed that this model can be used to test novel therapeutics by using a small molecule kinase inhibitor (fostamatinib) that rapidly attenuated both glomerular and pulmonary injury over a 4-day treatment period. We believe that this enhanced model of MPO-AAV will prove useful for the study of glomerular leucocyte behaviour and novel therapeutics in AAV in the future. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Abstract 91: FLT3 is a major driver of glucose consumption in squamous cell lung cancer

Abstract Squamous cell lung cancer (SqCLC) represents 30% of all cases of non-small cell lung cancer. Despite the prevalence of SqCLC, there are few molecularly targeted therapies for SqCLC patients. There is an urgent need for new approaches in this area. SqCLC cells consume elevated levels of glucose and are highly dependent on glucose consumption for their survival. Targeting glucose consumption may be a valuable therapeutic strategy in SqCLC but is challenged by the fact that glucose consumption is critical for vital organs. Our ability to selectively target glucose consumption in SqCLC is limited by an incomplete understanding of the shared and distinct mechanisms through which SqCLC and healthy cells drive glucose consumption. We recently developed and validated a high-throughput glucose consumption assay that serves as a platform technology for identifying new proteins and pathways that drive glucose consumption in cancerous and healthy cells. In this project, we screened H520, SK-MES-1, and H596 SqCLC cell lines against 3555 bioactive small molecules including a library of kinase inhibitors and FDA-approved drugs, and measured glucose consumption 24 hours after drug treatment. We discovered and validated 62 compounds that decreased glucose consumption per cell by &amp;gt;50% in at least one cell line. From this list, we searched for compounds that decreased glucose consumption in all three cell lines, by &amp;gt;70% in at least one cell line, and with an EC50 value &amp;lt;~1 µM; that have been used in mice and people; that have not been previously linked to glucose consumption; and that do not significantly decrease cell growth at the time point measured (24 hours post-treatment). This led us to further investigate Pacritinib, a small molecule kinase inhibitor in clinical trials for the treatment of myelofibrosis. Pacritinib reduces glucose consumption in all three SqCLC cell lines with an EC50 value of 1.2 µM in cell culture and blocks glucose consumption in cell line xenografts in vivo without affecting glucose consumption in healthy tissues. In the SqCLC cell lines, Pacritinib decreases protein levels of Hexokinase 1 and Hexokinase 2 as well as hexokinase activity as measured by a FRET-based glucose sensor. Hexokinase 1 overexpression in the SqCLC cell lines blocks Pacritinib from inhibiting glucose consumption. Pacritinib targets JAK2, TYK2, FLT3, and additional kinases at low nanomolar concentrations in vitro. Additional small molecule inhibitors of JAK2 and TYK2 had no effect on SqCLC glucose consumption. However additional inhibitors of FLT3 blocked glucose consumption in all three SqCLC cell lines. FLT3 overexpression increased SqCLC glucose consumption and blocked Pacritinib from inhibiting glucose consumption. In conclusion, using our high-throughput technology, we discovered that FLT3 is a selective and targetable driver of glucose consumption in SqCLC. Our data suggest that FLT3 activates glucose consumption by increasing Hexokinase 1 levels. Citation Format: Chiara Ghezzi, Robert Damoiseaux, Peter M. Clark. FLT3 is a major driver of glucose consumption in squamous cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 91.

Abstract 1387: MET inhibition radiosensitizes KRAS-mutant rectal cancer

Abstract Rectal carcinoma, representing about a third of all newly diagnosed colorectal cancers, is one of the most common malignant tumors. The standard of care for locally advanced rectal cancer (LARC), consisting of neoadjuvant chemo/radiotherapy prior to surgical resection, is poorly effective, leading to complete tumor regression only in 10-30% of the cases. Approximately 40% of LARC harbor activating KRAS mutations, which have been extensively associated with primary resistance to targeted EGFR therapy and with radioresistance as well. Previous work showed that the MET receptor tyrosine kinase supports radioresistance and can be inhibited to radiosensitize tumor cell subpopulations retaining stem-like properties. Here, we show that LARC often express high levels of MET and can be successfully radiosensitized by MET inhibition. This was assessed in rectal stem-like cells isolated from human tumors (rectospheres), and transplanted in the mouse to regenerate tumors that faithfully reproduce the phenotype, the genotype and the therapeutic response of the original tumor. Mechanistically, we found that radioresistant KRAS-mutant rectospheres display significantly higher basal levels of RAD51, a master regulator of DNA homologous recombination repair, and increased RAD51 recruitment to irradiation-induced DNA double-strand breaks, as compared with KRAS-wild type rectospheres. Importantly, we showed that MET pharmacological inhibition by small-molecule kinase inhibitors, combined with radiotherapy, impairs RAD51 expression and function. This leads to DNA damage accumulation and results in effective radiosensitization of K-RAS-mutant rectal stem-like cells in vitro and in vivo, and significant inhibition of experimental tumors. Therefore, preclinical evidence is provided that MET can be exploited as a therapeutic target to radiosensitize KRAS-mutant rectal cancer at stem-like cell level. Citation Format: Antonio D'Ambrosio, Federica Verginelli, Francesca Orzan, Raffaella Albano, Elena Casanova, Paolo Luraghi, Francesca De Bacco, Andrea Bertotti, Livio Trusolino, Rebecca Senetta, Anna Sapino, Elena Del Mastro, Marco Gatti, Paolo M. Comoglio, Carla Boccaccio. MET inhibition radiosensitizes KRAS-mutant rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1387.

Abstract 2182: Evaluation of somatic and germline mutations in ampullary carcinoma reveals actionable targets in multiple signaling pathways

Abstract Background: Ampullary carcinoma (AC) is a rare gastrointestinal cancer with few effective standard treatment options. For patients with unresectable diseases, the response rates for radiotherapy, chemotherapy, and chemoradiotherapy are low. Evaluation of the actionable mutations through genomic profiling will enable patients to enroll in matched clinical trials. Methods: To characterize actionable targets in 27 Chinese AC patients, a 500-gene next-generation sequencing panel was applied to assess the mutational status of their tumor tissues, including SNV, insertions/deletions, CNV and re-arrangements. Also, paired genomic DNA sample was extracted and analyzed by a 93-gene NGS panel to characterize germline variants. Results: Recurrent somatic mutated genes were KRAS (63%), TP53 (59%), APC (26%), SMAD4 (19%), CDKN2A (15%), ELF3 (11%), ERBB2 (11%), ERBB3 (11%) and MAP2K4 (11%). Consistent with previous studies, all mutations in AC driver gene ELF3 (n =3) were inactivating (three indels and one in splicing-site). The median tumor mutation burden (TMB) of our cohort was 1.6 (0-6.1) mutations/Mb and none of our patients had high microsatellite instability (MSI-H). Overall, we observed a wide spectrum of hotspot mutations in cancer driver genes including ERBB3 (n =2), FBXW7 (n =2), U2AF1 (n =2), BRAF (n =1), ERBB2/HER2 (n =1) and PIK3CA (n =1). The HER2 R678Q mutation, located in the transmembrane domain (TMD), activated HER2 signaling by improving the active dimer interface or stabilizing an activating conformation of HER2. Interestingly, both ERBB3 mutations (G284R and D297Y) were located in the extracellular domain (ECD) and their oncogenic activity also depended on the kinase-activity of ERBB2. These three mutations, together with one case of ERBB2 amplification, can be targeted by anti-HER2 antibodies and small-molecule HER2 kinase inhibitors. The BRAF N581I mutation is resistant to BRAF inhibitor vemurafenib but sensitive to MEK inhibitor trametinib. The PIK3CA N1044K mutation is oncogenic but its sensitivity to PIK3CA inhibitor alpelisib is unknown. Additionally, one patient had high level of FGFR2 amplification which may be suitable for the clinical trials of FGFR2 inhibitors pemigatnib and erdafitnib. Furthermore, two patients carried loss-of-function germline mutations in DNA repair genes PALB2 and CHEK2 which may confer synthetic lethality with PARP inhibitors. Taken together, at least eight (29.6%) patients in our cohort had actionable therapeutic targets. Conclusions: Through comprehensive genomic characterization of Chinese AC patients, we identified multiple actionable mutations in multiple signaling pathways. Our findings indicated that molecular profiling can provide clinical benefits to a significant portion of AC patients. Citation Format: Fei Wang, Si Liu, Xiaomo Li, Zhiming Zhao, Tonghui Ma, Hongling Yuan, Rong Liu. Evaluation of somatic and germline mutations in ampullary carcinoma reveals actionable targets in multiple signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2182.

Abstract LB132: CDK8/19 inhibition overcomes in vitro and in vivo resistance to lapatinib in HER2+ breast cancer via STAT1 and STAT3

Abstract One of every five breast cancers are driven by gene amplification and overexpression of HER2/ERBB2 tyrosine kinase. HER2+ breast cancers are treated both by monoclonal antibodies (such as trastuzumab) and small-molecule HER2 kinase inhibitors (such as lapatinib). However, some HER2+ tumors are inherently resistant to HER2-targeting drugs and most of metastatic cancers treated with such drugs eventually acquire resistance. We have analyzed the effect of small-molecule inhibitors of transcriptional serine/threonine kinases CDK8 and CDK19, paralogs of the Mediator kinase that modulates the activity of several signal-responsive transcription factors, on the response to lapatinib in a panel of HER2+ breast cancer cell lines. Selective CDK8/19 inhibitors senexin B and SNX631 showed in vitro synergy with lapatinib in different lapatinib-responsive cell lines and overcame lapatinib resistance in cells with either inherent or acquired resistance to this drug. In vivo treatment with SNX631 significantly inhibited the growth of cell-line based and a patient-derived xenograft (PDX) models of HER2+ breast cancer. A combination of SNX631 and lapatinib drastically suppressed the growth of tumors formed by lapatinib-sensitive HCC1954 cells and their lapatinib-resistant derivative HCC1954-L, overcoming lapatinib resistance of the latter. GSEA analysis of RNA-Seq data from cell treated in vitro with lapatinib, senexin B or their combination, showed that lapatinib activated the JAK/STAT pathway but the addition of the CDK8/19 inhibitor suppressed such induction. CDK8/19 inhibitors, when combined with lapatinib, suppressed STAT3 and STAT1 phosphorylation at Ser727. CRISPR/CAS9 knockout of STAT1 and STAT3 together (but not individually) sensitized HER2+ breast cancer cells to lapatinib and reduced the lapatinib-sensitizing effect of CDK8/19 inhibitors, suggesting that STAT1 and STAT3 mediate the effect of CDK8/19. Through miRNA-Seq and QPCR analysis, we have also identified two miRNAs that are regulated by STAT1 or STAT3 and overexpressed in lapatinib-resistant cells. These miRNAs are upregulated upon lapatinib treatment, whereas the combination of lapatinib with senexin B decreases their expression, suggesting that these miRNAs may mediate the effects of CDK8/19 and STAT1/3 on lapatinib resistance. Our results suggest that combining HER2- and CDK8/19-targeting drugs could be a promising therapeutic direction for HER2+ breast cancers after the failure of HER2-targeted therapy. Citation Format: Xiaokai Ding, Amanda C. Sharko, Martina S-J McDermott, Hao Ji, Alexander Chumanevich, Gary P. Schools, Zachary T. Mack, Elena Pugacheva, Victor Tatarskiy, Anastasia Khrustaleva, Alexander Tyakht, Michael Shtutman, Mengqian Chen, Igor Roninson, Eugenia V. Broude. CDK8/19 inhibition overcomes in vitro and in vivo resistance to lapatinib in HER2+ breast cancer via STAT1 and STAT3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB132.