Small-molecule Kinase Inhibitors Research Articles

Discovery of Orally Bioavailable FGFR2/FGFR3 Dual Inhibitors via Structure-Guided Scaffold Repurposing Approach.

Fibroblast growth factor receptors (FGFRs) are transmembrane receptor tyrosine kinases that regulate multiple physiological processes. Aberrant activation of FGFR2 and FGFR3 has been linked to the pathogenesis of many tumor types, including cholangiocarcinoma and bladder cancer. Current therapies targeting the FGFR2/3 pathway exploiting small-molecule kinase inhibitors are associated with adverse events due to undesirable inhibition of FGFR1 and FGFR4. Isoform-specific FGFR2 and FGFR3 inhibitors that spare FGFR1 and FGFR4 could offer a favorable toxicity profile and improved therapeutic window to current treatments. Herein we disclose the discovery of dual FGFR2/FGFR3 inhibitors exploiting scaffold repurposing of a previously reported ALK2 tool compound. Structure-based drug design and structure-activity relationship studies were employed to identify selective and orally bioavailable inhibitors with equipotent activity toward wild-type kinases and a clinically observed gatekeeper mutant.

2-Ethynylbenzaldehyde-Based, Lysine-Targeting Irreversible Covalent Inhibitors for Protein Kinases and Nonkinases.

Lysine-targeting irreversible covalent inhibitors have attracted growing interests in recent years, especially in the fields of kinase research. Despite encouraging progress, few chemistries are available to develop inhibitors that are exclusively lysine-targeting, selective, and cell-active. We report herein a 2-ethynylbenzaldehyde (EBA)-based, lysine-targeting strategy to generate potent and selective small-molecule inhibitors of ABL kinase by selectively targeting the conserved catalytic lysine in the enzyme. We showed the resulting compounds were cell-active, capable of covalently engaging endogenous ABL kinase in K562 cells with long-residence time and few off-targets. We further validated the generality of this strategy by developing EBA-based irreversible inhibitors against EGFR (a kinase) and Mcl-1 (a nonkinase) that covalently reacted with the catalytic and noncatalytic lysine within each target.

Understanding the Risk of Drug Interactions Between Ritonavir-Containing COVID-19 Therapies and Small-Molecule Kinase Inhibitors in Patients With Cancer.

The introduction of COVID-19 therapies containing ritonavir has markedly expanded the scope of use for this medicine. As a strong cytochrome P450 3A4 inhibitor, the use of ritonavir is associated with a high drug interaction risk. There are currently no data to inform clinician regarding the likely magnitude and duration of interaction between ritonavir-containing COVID-19 therapies and small-molecule kinase inhibitors (KIs) in patients with cancer. Physiologically based pharmacokinetic modeling was used to conduct virtual clinical trials with a parallel group study design in the presence and absence of ritonavir (100 mg twice daily for 5 days). The magnitude and time course of changes in KI exposure when coadministered with ritonavir was evaluated as the primary outcome. Dosing of ritonavir resulted in a > 2-fold increase in steady-state area under the plasma concentration-time curve and maximal concentration for six of the 10 KIs. When the KI was coadministered with ritonavir, dose reductions to between 10% and 75% of the original dose were required to achieve an area under the plasma concentration-time curve within 1.25-fold of the value in the absence of ritonavir. To our knowledge, this study provides the first data to assist clinicians' understanding of the drug interaction risk associated with administering ritonavir-containing COVID-19 therapies to patients with cancer who are currently being treated with KIs. These data may support clinicians to make more informed dosing decisions for patients with cancer undergoing treatment with KIs who require treatment with ritonavir-containing COVID-19 antiviral therapies.

RON Receptor Tyrosine Kinase in Tumorigenic Stemness as a Therapeutic Target of Antibody-drug Conjugates for Eradication of Triple-negative Breast Cancer Stem Cells.

Cancer stem-like cells in triple-negative breast cancer (TNBC-SLCs) are the tumorigenic core for malignancy. Aberrant expression of the RON receptor tyrosine kinase has implications in TNBC tumorigenesis and malignancy. In this study, we identified the RON receptor as a pathogenic factor contributing to TNBC cell stemness and validated anti-RON antibody-drug conjugate Zt/g4-MMAE for eradication of RONexpressing TNBC-SLCs. Immunofluorescence and Western blotting were used for analyzing cellular marker expression. TNBC-SLCs were isolated by magnetic-immunofluorescence cell-sorting techniques. Spheroids were generated using the ultralow adhesion culture methods. Levels of TNBC-SLC chemosensitivity were determined by MTS assays. TNBC-SLC mediated tumor growth was determined in athymic nude mice. The effectiveness of Zt/g4-induced RON internalization was measured by immunofluorescence analysis. Efficacies of Zt/g4-MMAE in killing TNBC-SLCs in vitro and in eradicating TNBC-SLCmediated tumors were determined in mouse models. All data were statistically analyzed using the GraphPad Prism 7 software. Increased RON expression existed in TNBC-SLCs with CD44+/CD24- phenotypes and ALDH activities and facilitated epithelial to mesenchymal transition. RON-positive TNBC-SLCs enhanced spheroid-formatting capability compared to RON-negative TNBC-SLCs, which were sensitive to small molecule kinase inhibitor BMS-777607. Increased RON expression also promoted TNBC-SLC chemoresistance and facilitated tumor growth at an accelerated rate. In vitro, Zt/g4-MMAE caused massive TNBC-SLC death with an average IC50 value of ~1.56 μg per/ml and impaired TNBC cell spheroid formation. In mice, Zt/g4-MMAE effectively inhibited and/or eradicated TNBC-SLC mediated tumors in a single agent regimen. Sustained RON expression contributes to TNBC-SLC tumorigenesis. Zt/g4-MMAE is found to be effective in vivo in killing TNBC-SLC-mediated xenograft tumors. Our findings highlight the feasibility of Zt/g4-MMAE for the eradication of TNBC-SLCs in the future.

Small Molecule EGFR Inhibitors as Anti-Cancer Agents: Discovery, Mechanisms of Action, and Opportunities.

Epidermal growth factor receptors (EGFRs) are a class of receptor tyrosine kinase that are also called ErbB1 and HER1. EGFR tyrosine kinase activity inhibition is considered a promising therapeutic strategy for the treatment of cancer. Many small-molecule inhibitors of EGFR tyrosine kinase (EGFR-TK), from medicinally privileged molecules to commercial drugs, have been overviewed. Particular attention has been paid to the structure of the molecule and its mechanism of action if reported. Subsequent classification of the molecules under discussion has been carried out. Both natural and synthetic and reversible and irreversible EGFR-tyrosine kinase inhibitors have been discussed. Various types of cancers that are caused by overexpression of the EGFR gene, their possible molecular origins, and their natures have also been counted in this article. Because the EGFR signaling pathway controls the proliferation, growth, survival, and differentiation of cells, and the mutated EGFR gene overproduces EGFR protein, which ultimately causes several types of cancer, proper understanding of the molecular dynamics between the protein structure and its inhibitors will lead to more effective and selective EGFR-TKIs, which in turn will be able to save more lives in the battle against cancer.

Futibatinib (Lytgobi) for cholangiocarcinoma

STRUCTURE: Futibatinib (brand name LytgobiTM) is a small molecule kinase inhibitor with the molecular formula C22H22N6O3 and a molecular mass of 418.45 g/mole. The drug is a pyrazolo[3,4-d]pyrimidine derivative with an attached α,β-unsaturated amide warhead, also called a Michael acceptor, that forms a covalent bond with a cysteine residue in the fibroblast growth factor receptor (FGFR) protein-tyrosine kinase glycine-rich loop.

Quantitation of osimertinib, alectinib and lorlatinib in human cerebrospinal fluid by UPLC-MS/MS

Overall survival in metastatic lung cancer has been dramatically improved with the use of small molecule kinase inhibitors (SMKIs). Quantification of SMKI in cerebrospinal fluid (CSF) can be used to assess penetration of these drugs into the central nervous system. This paper describes an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for quantification of the SMKIs alectinib, lorlatinib and osimertinib in human CSF. Alectinib-d8 and dasatinib-d8 were used as internal standards. Aliquots with 25 µL CSF/30% albumin (9:1,v/v) were mixed with 100 µL internal standard solution consisting of 1 ng/mL dasatinib-d8 and alectinib-d8 in acetonitrile. The analytes were separated by an Acquity UPLC® HSS T3 column (2.1 ×150 mm, 1.8 µm), using gradient elution (ammonium formate pH 4.5, acetonitrile) with a flow rate of 0.400 mL/min. All calibration curves were linear for the concentration range from 2.50 to 250 ng/mL. Within-run and between-run precision varied from 0.72% to 11.7%, with accuracy ranging from 95.3% to 113.2%. For all compounds, a high degree of non-specific binding to the vacutainer was observed. This issue could be countered easily by a combination of pre-coating with BSA solution (30%) in phosphate buffer pH 4.2, and immediate sample mixture with BSA solution after collection. To test the clinical applicability, CSF was collected in seven unique patients using alectinib (n = 1), lorlatinib (n = 2), and osimertinib (n = 4). Measured CSF trough concentrations ranged between 3.37 and 116 ng/mL.

NanoBRET™ Live-Cell Kinase Selectivity Profiling Adapted for High-Throughput Screening.

Kinases represent one of the most therapeutically tractable targets for drug discovery in the twenty-first century. However, confirming engagement and achieving intracellular kinase selectivity for small-molecule kinase inhibitors can represent noteworthy challenges. The NanoBRETTM platform enables broad-spectrum live-cellkinase selectivity profiling in most laboratory settings, without advanced instrumentation or expertise. However, the prototype workflow for this selectivity profiling is currently limited to manual liquid handling and 96-well plates. Herein, we describe a scalable workflow with automation and acoustic dispensing, thus dramatically improving the throughput. Such adaptations enable profiling of larger compound sets against 192 full-length protein kinases in live cells, with statistical robustness supporting quantitative analysis.

Calcium signaling induced by 15-deoxy-prostamide-J2 promotes cell death by activating PERK, IP3R, and the mitochondrial permeability transition pore.

Melanoma is the deadliest form of skin cancer in the US. Although immunotherapeutic checkpoint inhibitors and small-molecule kinase inhibitors have dramatically increased the survival of patients with melanoma, new or optimized therapeutic approaches are still needed to improve outcomes. 15-deoxy-Δ12,14-prostamide J2 (15d-PMJ2) is an investigational small-molecule that induces ER stress-mediated apoptosis selectively in tumor cells. Additionally, 15d-PMJ2 reduces melanoma growth in vivo. To assess the chemotherapeutic potential of 15d-PMJ2, the current study sought to uncover molecular pathways by which 15d-PMJ2 exerts its antitumor activity. B16F10 melanoma and JWF2 squamous cell carcinoma cell lines were cultured in the presence of pharmacological agents that prevent ER or oxidative stress as well as Ca2+ channel blockers to identify mechanisms of 15d-PMJ2 cell death. Our data demonstrated the ER stress protein, PERK, was required for 15d-PMJ2-induced death. PERK activation triggered the release of ER-resident Ca2+ through an IP3R sensitive pathway. Increased calcium mobilization led to mitochondrial Ca2+ overload followed by mitochondrial permeability transition pore (mPTP) opening and the deterioration of mitochondrial respiration. Finally, we show the electrophilic double bond located within the cyclopentenone ring of 15d-PMJ2 was required for its activity. The present study identifies PERK/IP3R/mPTP signaling as a mechanism of 15d-PMJ2 antitumor activity.

Combined inhibition of ACLY and CDK4/6 reduces cancer cell growth and invasion.

The use of small molecule kinase inhibitors, which target specific enzymes that are overactive in cancer cells, has revolutionized cancer patient treatment. To treat some types of breast cancer, CDK4/6 inhibitors, such as palbociclib, have been developed that target the phosphorylation of the retinoblastoma tumor suppressor gene. Acquired resistance to CDK4/6 inhibitors may be due to activation of the AKT pro‑survival signaling pathway that stimulates several processes, such as growth, metastasis and changes in metabolism that support rapid cell proliferation. The aim of the present study was to investigate whether targeting ATP citrate lyase (ACLY), a downstream target of AKT, may combine with CDK4/6 inhibition to inhibit tumorigenesis. The present study determined that ACLY is activated in breast and pancreatic cancer cells in response to palbociclib treatment and AKT mediates this effect. Inhibition of ACLY using bempedoic acid used in combination with palbociclib reduced cell viability in a panel of breast and pancreatic cancer cell lines. This effect was also observed using breast cancer cells grown in 3D cell culture. Mechanistically, palbociclib inhibited cell proliferation, whereas bempedoic acid stimulated apoptosis. Finally, using Transwell invasion assays and immunoblotting, the present study demonstrated that ACLY inhibition blocked cell invasion, when used alone or in combination with palbociclib. These data may yield useful information that could guide the development of future therapies aimed at the reduction of acquired resistance observed clinically.

Kinase Inhibitors in the Treatment of Ovarian Cancer: Current State and Future Promises.

Ovarian cancer is the deadliest gynecological cancer, the high-grade serous ovarian carcinoma (HGSC) being its most common and most aggressive form. Despite the latest therapeutical advancements following the introduction of vascular endothelial growth factor receptor (VEGFR) targeting angiogenesis inhibitors and poly-ADP-ribose-polymerase (PARP) inhibitors to supplement the standard platinum- and taxane-based chemotherapy, the expected overall survival of HGSC patients has not improved significantly from the five-year rate of 42%. This calls for the development and testing of more efficient treatment options. Many oncogenic kinase-signaling pathways are dysregulated in HGSC. Since small-molecule kinase inhibitors have revolutionized the treatment of many solid cancers due to the generality of the increased activation of protein kinases in carcinomas, it is reasonable to evaluate their potential against HGSC. Here, we present the latest concluded and on-going clinical trials on kinase inhibitors in HGSC, as well as the recent work concerning ovarian cancer patient organoids and xenograft models. We discuss the potential of kinase inhibitors as personalized treatments, which would require comprehensive assessment of the biological mechanisms underlying tumor spread and chemoresistance in individual patients, and their connection to tumor genome and transcriptome to establish identifiable subgroups of patients who are most likely to benefit from a given therapy.

Protein Kinase Inhibitors as a New Target for Immune System Modulation and Brain Cancer Management

High-grade brain tumors are malignant tumors with poor survival and remain the most difficult tumors to treat. An important contributing factor to the development and progression of brain tumors is their ability to evade the immune system. Several immunotherapeutic strategies including vaccines and checkpoint inhibitors have been studied to improve the effectiveness of the immune system in destroying cancer cells. Recent studies have shown that kinase inhibitors, capable of inhibiting signal transduction cascades that affect cell proliferation, migration, and angiogenesis, have additional immunological effects. In this review, we explain the beneficial therapeutic effects of novel small-molecule kinase inhibitors and explore how, through different mechanisms, they increase the protective antitumor immune response in high-grade brain tumors.

Kinase inhibitors for cancer alter metabolism, blood glucose, and insulin.

Small molecule kinase inhibitors (SMKIs) are a class of therapeutic drugs that target protein kinases in diseases such as cancer. SMKIs are often designed to inhibit kinases involved in cell proliferation, but these drugs alter cell metabolism and the endocrine control of organismal metabolism. SMKI treatment in diabetic cancer patients reveals that certain SMKIs improve blood glucose levels and can mitigate insulin dependence or diabetic medication requirements in both type 1 diabetes (T1D) and type 2 diabetes (T2D). Certain SMKIs can preserve functional β-cell mass and increase insulin secretion or insulin sensitivity. It is not yet clear why different SMKIs can have opposing effects on insulin and blood glucose. Understanding the therapeutic effects of these drugs in T1D and T2D is complicated by overlapping off-target effects of SMKIs. The potency of inhibition of the intended protein kinase and inhibition of multiple off-target kinases may underpin conflicting reports of how certain SMKIs alter blood glucose and insulin. We summarize the effects of SMKIs on the intended and off-target kinases that can alter blood glucose and insulin, including c-Abl, c-Kit, EGFR, and VEGF. Inhibition of PDGFRβ consistently lowers blood glucose in T1D and T2D. The effects of SMKIs on the kinases that regulate immune pathways, such as BTK and RIPKs, mediate many of the diverse effects of these drugs on metabolism. We highlight that inhibition of RIPK2 by SMKIs is a central node in metabolism that influences key metabolic pathways including lipolysis, blood glucose control, insulin secretion, and insulin resistance.

Recent Research Advances in Small-Molecule Pan-PIM Inhibitors

PIM kinase is consequently emerging as a promising target for cancer therapeutics and immunomodulation. PIM kinases are overexpressed in a variety of hematological malignancies and solid tumors, and their inhibition has become a strong therapeutic interest. Currently, some pan-PIM kinase inhibitors are being developed under different phases of clinical trials. Based on the different scaffold structures, they can be classified into various subclasses. The X-ray structure of the kinase complex outlines the rationale of hit compound confirmation in the early stage. Structure–activity relationships allow us to rationally explore chemical space and further optimize multiple physicochemical and biological properties. This review focuses on the discovery and development of small-molecule pan-PIM kinase inhibitors in the current research, and hopes to provide guidance for future exploration of the inhibitors.

KID: A Kinase-Focused Interaction Database and Its Application in the Construction of Kinase-Focused Molecule Databases.

Protein kinases are important drug targets for the treatment of several diseases. The interaction between kinases and ligands is vital in the process of small-molecule kinase inhibitor (SMKI) design. In this study, we propose a method to extract fragments and amino acid residues from crystal structures for kinase-ligand interactions. In addition, core fragments that interact with the important hinge region of kinases were extracted along with their decorations. Based on the superimposed structural data of kinases from the kinase-ligand interaction fingerprint and structure database, we obtained two libraries, namely, a hinge-unfocused fragment-amino acid pair library (FAP Lib) that contains 6672 pairs of fragments and corresponding amino-acids, and a hinge-focused hinge binder library (HB Lib) of 3560 pairs of hinge-binding scaffolds with their corresponding decorations. These two libraries constitute a kinase-focused interaction database (KID). In depth analysis was conducted on KID to explore important characteristics of fragments in the design of SMKIs. With KID, we built two kinase-focused molecule databases, one called Recomb_DB, which contains 1,72,346 molecules generated through fragment recombination based on the FAP Lib, and another called RsdHB_DB, which contains 93,030 molecules generated based on our HB Lib using molecular generation methods. Compared with five databases both commercial and non-commercial, these two databases both ranked top 3 in scaffold diversity, top 4 in molecule fingerprint diversity, and are more focused on the chemical space of kinase inhibitors. Hence, KID presents a useful addition to existing databases for the exploration of novel SMKIs.

Liposomal amphotericin B-the future.

Advances in medicine have led to a growing number of people with compromised or suppressed immune systems who are susceptible to invasive fungal infections. In particular, severe fungal infections are becoming increasingly common in ICUs, affecting people within and outside of traditional risk groups alike. This is exemplified by the emergence of severe viral pneumonia as a significant risk factor for invasive pulmonary aspergillosis, and the recognition of influenza-associated pulmonary aspergillosis and, more recently, COVID-19-associated pulmonary aspergillosis. The treatment landscape for haematological malignancies has changed considerably in recent years, and some recently introduced targeted agents, such as ibrutinib, are increasing the risk of invasive fungal infections. Consideration must also be given to the risk of drug-drug interactions between mould-active azoles and small-molecule kinase inhibitors. At the same time, infections caused by rare moulds and yeasts are increasing, and diagnosis continues to be challenging. There is growing concern about azole resistance among both moulds and yeasts, mandating continuous surveillance and personalized treatment strategies. It is anticipated that the epidemiology of fungal infections will continue to change and that new populations will be at risk. Early diagnosis and appropriate treatment remain the most important predictors of survival, and broad-spectrum antifungal agents will become increasingly important. Liposomal amphotericin B will remain an essential therapeutic agent in the armamentarium needed to manage future challenges, given its broad antifungal spectrum, low level of acquired resistance and limited potential for drug-drug interactions.

Targeting Gatekeeper Mutations for Kinase Drug Discovery.

Clinically acquired resistance is a major challenge in cancer therapies with small-molecule kinase inhibitors (SMKIs). Gatekeeper mutations in the ATP-binding pocket of kinases are the most common mutations leading to acquired resistance. To date, seven new-generation kinase inhibitors targeting gatekeeper mutations have been approved by the FDA; however, the clinical need is still unmet. Here, we systematically summarize the types of gatekeeper mutations across the kinase family, the structural basis for acquired resistance, and newly developed SMKIs targeting gatekeeper mutations as well as highlight the opportunities and challenges of kinase drug discovery for targeting gatekeeper mutations.

Preliminary Results of a Phase-2 Clinical Trial of the ALK-2 Inhibitor Ker-047 for Treatment of Iron Refractory Iron Deficiency Anemia

Background: Iron refractory iron deficiency anemia (IRIDA) is a rare autosomal recessive disorder, caused by a genetic defect in TMPRSS6. This results in increased signaling through the bone morphogenic protein (BMP)/sons of mothers against decapentaplegic (SMAD) pathway, leading to disproportionally high levels of hepcidin relative to body iron status. Hepcidin reduces iron transport to the plasma by degrading the cellular iron exporter ferroportin, resulting in systemic iron deficiency and microcytic anemia. Symptomatic interventions such as intravenous iron administration results in iron sequestration in the reticular endothelial system (RES) and generally only a partial response of hemoglobin concentrations. Further, the potential long-term adverse effects of chronic intravenous iron therapy and associated elevated RES-iron levels are unknown in these patients. Blood transfusion can be administered in cases of severe symptomatic anemia but is generally avoided due to transfusion-related risks. Currently, there is an unmet need for more effective, safe, convenient, and direct-acting treatments for IRIDA. Given the central role of aberrant BMP/SMAD signaling in hepcidin production, selective inhibition of the BMP receptor ALK2 to reduce hepcidin represents a rational approach to target a precipitating factor of IRIDA. Preclinical studies in the TMPRSS6 siRNA knockdown mouse model of IRIDA demonstrated that treatment with a selective ALK2 inhibitor reduced hepcidin, increased serum iron, and ameliorated anemia. KER-047 is a novel, oral, investigational, small-molecule kinase inhibitor with potent and selective inhibitory activity against the ALK2 receptor. In a Phase 1 clinical trial, single-ascending and multiple-ascending doses of KER-047 administered to healthy participants resulted in robust decreases from baseline in hepcidin levels, increases in serum iron and transferrin saturation (TSAT), decreases in serum ferritin, and increases in reticulocyte hemoglobin content (Ret-He). Based on these observations, KER-047 was advanced to Phase 2 clinical trials. Aims: The primary objective of this ongoing Phase 2 clinical trial is to evaluate the safety and tolerability of ascending doses of KER-047 in participants with IRIDA. Secondary objectives include evaluation of pharmacokinetic (PK) and pharmacodynamic (PD) parameters. Methods: This is a 2-part open label, dose escalation and dose expansion trial. Part 1 (dose escalation) consists of up to 4 ascending dose cohorts starting with 25 mg q.d. Part 2 will be the dose expansion phase with safety as the primary focus. Patients diagnosed with IRIDA may enroll in all cohorts in Parts 1 and 2. Key inclusion criteria include diagnosis of IRIDA based on a homozygous or compound heterozygous TMPRSS6 genotype, a TSAT <15%, a ferritin level 50-700 µg/L and hemoglobin <13.8 g/dL (males) or <12.7g/dL (females) and signed informed consent. Patients are excluded if they have received IV iron treatment within 28 days prior to trial entry. Participants will be treated once daily with KER-047 for a 2-week period, followed by a 2-week washout period. Data are analyzed by using descriptive statistics. Results: At the time of this abstract submission, one participant had enrolled in the first dose-expansion cohort (KER-047 25mg q.d.). This participant reported intermittent dizziness, however no serious adverse events were observed during treatment. Hemoglobin concentration and mean corpuscular volume remained stable. Ret-He increased by day 5 of treatment. Ferritin levels decreased during treatment and returned to baseline levels after the 2-week treatment period. TSAT/hepcidin ratio, which is characteristically below reference intervals in IRIDA patients, was slightly increased but returned to baseline levels after 2-weeks treatment. Conclusion: Data are preliminary and limited to a single participant thus far in whom KER-047 25mg q.d. was well tolerated. Changes in red cell and iron parameters at this low dose support development of KER-047 as an agent to potentially address an underlying cause of IRIDA. At the time of this abstract submission, two additional patients are scheduled for screening to complete the first dose cohort. We anticipate data from the total of three patients in time for the presentation to further characterize safety of KER-047 and substantiate effects on iron metabolism. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Super-Enhancer-Driven PIM Kinase Upregulation in Multiple Myeloma Maintains the Plasma Cell-Specific Oncogenic and Microenvironmental Circuits, and Can be Efficiently Targeted By the Pan-PIM Inhibitor MEN1703

The three constitutively active PIM serine/threonine kinases have been previously reported to initiate and sustain lymphoid oncogenesis. In subsequent CRISPR/Cas9 drop-out screens, PIM kinases were highlighted as critical oncogenes in MM, which led to clinical trials of small-molecule PIM kinase inhibitors. Surprisingly, PIM inhibitors in MM patients exhibited limited activity due to their cytostatic, rather cytotoxic effects. These clinical trial results prompted us to further investigate the consequences of PIM kinase inhibition in MM using recently developed pan-PIM/FLT3 inhibitor MEN1703. Immunohistochemical staining revealed a markedly increased PIM1/2/3 levels in malignant plasma cells in comparison with normal plasma cells from healthy donors, with particularly abundant PIM2 expression. Through the integration of CRISPR/Cas9 dropout screen and genome-wide H3K27Ac profiling in MM cells, we identified PIM2 as a super-enhancer-driven oncogene in MM. BET inhibitor JQ1, which disrupts super-enhancer assembly, downregulated PIM kinase expression. Since super-enhancer associated genes are often the most essential nodes of key cellular pathways to be potentially targeted, these results further underscore the critical role of PIMs in MM. Next, we compared the activity of MEN1703 with all available clinical-stage PIM inhibitors in a panel of >20 different cell lines and found almost universal resistance to 4 out of 5 pan-PIM inhibitors administered at submicromolar concentrations achievable in vivo (Fig. 1A). In contrast, MEN1703 exhibited universal cytotoxic activity. We confirmed these results using patient-derived multiple myeloma cells cultured ex vivo and genetic knockdowns of PIM kinases. Mechanistically, MEN1703 decreased the activity of mTOR, JAK/STAT, and NFκB pathways essential for supporting myeloma cell survival. Consequently, we observed diminished expression of MYC and IRF4, two transcription factors crucial for supporting plasma cell-specific oncogenic program. In the IHC analysis of PIMs expression in MM tissue microarray, we also noted a distinctly high expression of PIM3 in myeloma-associated endothelial cells (ECs), but not in healthy bone marrow ECs. Since ECs play a significant role in shaping the bone marrow microenvironment and directly support the dissemination and survival of plasma cells, we studied the mechanism of PIM3 overexpression, PIM3 function, and the consequences of its inhibition in MM ECs. We found that myeloma-secreted VEGFA is responsible for PIM induction in ECs and that endothelial cells reciprocally induce PIMs expression in MM cells in a paracrine manner, thus increasing their proliferation and inhibiting cell death. PIM inhibition blocked these effects, which was confirmed using cocultures with primary MM endothelial cells. PIM inhibition decreased the MM EC vessel formation by decreasing the activity of endothelial GTPase RhoA, which is responsible for actin cytoskeleton formation during angiogenesis and cytokine secretion. Collectively, our data demonstrate that PIM inhibition with MEN1703 induces MM cell death and abolishes important tumor cell-ECs interactions (Fig. 1B), thus representing a promising therapeutic target in this disease. The clinical utility of next generation/more potent PIM inhibitor in MM should be re-investigated. Study supported by National Science Centre Grants PRELUDIUM 2018/31/N/NZ5/03214, ETIUDA 2020/36/T/NZ5/00610 and Ministry of Science and Higher Education Diamond Grant DI2015 007145. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting EGR1-Medited Metabolic Reprogramming to Overcome Ibrutinib Resistance in Aggressive B Cell Lymphomas

Targeting specific signaling pathways in malignant cells through the use of small-molecule kinase inhibitors has revolutionized the treatment of malignancies. Bruton tyrosine kinase (BTK), a key component of the B cell receptor (BCR) signaling pathway, is a promising molecular target in several types of lymphoma, including diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL). Ibrutinib, a selective BTK inhibitor, has achieved initial response rates as high as 70% in these lymphomas when used as a single agent. Acquired drug resistance, however, is significant and together with primary resistance makes ibrutinib by itself unsuitable if the goal of treatment is long-term survival. Therefore, understanding and targeting ibrutinib resistance mechanisms is an unmet clinical need. DLBCL, which represents 30% to 40% of newly diagnosed lymphomas, comprises two main molecular subtypes: activated B cell-like (ABC) and germinal center B cell-like (GCB). The BCR/BTK signaling pathway is essential for the growth of ABC DLBCL, which is more aggressive and less curable than GCB DLBCL with current regimens. We established two representative ABC DLBCL cell lines (TMD8 and OCI-Ly10) with ibrutinib resistance by gradually increasing the concentration of ibrutinib during passage in culture. RNA-seq analysis demonstrated that the BCR pathway gene signature is enriched in resistant cell lines when compared to parental cells. The most upregulated gene is EGR1, a transcription factor that activates multiple oncogenic pathways including MYC and E2F. Elevated EGR1 expression is also observed in ibrutinib-resistant primary mantle cell lymphoma cells when treated with ibrutinib. Using multiple metabolic and genetic approaches, we discovered that overexpression of EGR1 causes metabolic reprogramming to oxidative phosphorylation (OXPHOS) and ibrutinib resistance. Mechanistically, EGR1 mediates metabolic reprogramming through transcriptional activation of PDP1, a phosphatase that dephosphorylates and activates the E1 component of the large pyruvate dehydrogenase complex. Therefore, EGR1-mediated PDP1 activation accelerates intracellular ATP production via the mitochondrial tricarboxylic acid (TCA) cycle, leading to sufficient energy to enhance the proliferation and survival of ibrutinib-resistant lymphoma cells. Finally, we demonstrate that targeting OXPHOS with IM156, a newly developed OXPHOS inhibitor, inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in patient-derived xenograft mouse models. These findings suggest that targeting EGR1-medited metabolic reprogramming to OXPHOS with IM156 provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory DLBCL or MCL.