Anti-SARS-CoV-2 activity of targeted kinase inhibitors: Repurposing clinically available drugs for COVID-19 therapy.

Coronavirus disease 2019 (COVID-19) remains a major public health concern, and vaccine unavailability, hesitancy, or failure underscore the need for discovery of efficacious antiviral drug therapies. Numerous approved drugs target protein kinases associated with viral life cycle and symptoms of infection. Repurposing of kinase inhibitors is appealing as they have been vetted for safety and are more accessible for COVID-19 treatment. However, an understanding of drug mechanism is needed to improve our understanding of the factors involved in pathogenesis. We tested the in vitro activity of three kinase inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including inhibitors of AXL kinase, a host cell factor that contributes to successful SARS-CoV-2 infection. Using multiple cell-based assays and approaches, gilteritinib, nintedanib, and imatinib were thoroughly evaluated for activity against SARS-CoV-2 variants. Each drug exhibited antiviral activity, but with stark differences in potency, suggesting differences in host dependency for kinase targets. Importantly, for gilteritinib, the amount of compound needed to achieve 90% infection inhibition, at least in part involving blockade of spike protein-mediated viral entry and at concentrations not inducing phospholipidosis (PLD), approached a clinically achievable concentration. Knockout of AXL, a target of gilteritinib and nintedanib, impaired SARS-CoV-2 variant infectivity, supporting a role for AXL in SARS-CoV-2 infection and supporting further investigation of drug-mediated AXL inhibition as a COVID-19 treatment. This study supports further evaluation of AXL-targeting kinase inhibitors as potential antiviral agents and treatments for COVID-19. Additional mechanistic studies are needed to determine underlying differences in virus response.

Essential role of the histone lysine demethylase KDM4A in the biology of malignant pleural mesothelioma (MPM).

BACKGROUND: Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a dismal prognosis. There is increasing interest in targeting chromatin regulatory pathways in difficult-to-treat cancers. In preliminary studies, we found that KDM4A (lysine-specific histone demethylase 4) was overexpressed in MPM. METHODS: KDM4A protein expression was determined by immunohistochemistry or immunoblotting. Functional inhibition of KDM4A by targeted knockdown and small molecule drugs was correlated to cell growth using cell lines and a xenograft mouse model. Gene expression profiling was performed to identify KDM4A-dependent signature pathways. RESULTS: Levels of KDM4A were found to be significantly elevated in MPM patients compared to normal mesothelial tissue. Inhibiting the enzyme activity efficiently reduced cell growth in vitro and reduced tumour growth in vivo. KDM4A inhibitor-induced apoptosis was further enhanced by the BH3 mimetic navitoclax. KDM4A expression was associated with pathways involved in cell growth and DNA repair. Interestingly, inhibitors of the DNA damage and replication checkpoint regulators CHK1 (prexasertib) and WEE1 (adavosertib) within the DNA double-strand break repair pathway, cooperated in the inhibition of cell growth. CONCLUSIONS: The results establish a novel and essential role for KDM4A in growth in preclinical models of MPM and identify potential therapeutic approaches to target KDM4A-dependent vulnerabilities.

Post-transplant maintenance therapy in patients with FLT3-mutated acute myeloid leukemia: Real-world treatment patterns and outcomes.

OBJECTIVES: Maintenance therapy is one strategy to prolong survival in patients with acute myeloid leukemia (AML) following hematopoietic stem cell transplantation (HSCT). We evaluated real-world treatment patterns and outcomes in patients with newly diagnosed FLT3-mutated AML receiving HSCT after complete remission with first-line chemotherapy. METHODS: A global, retrospective chart review to evaluate maintenance therapy and outcomes in patients with FLT3-mutated AML after HSCT. RESULTS: Data from 1208 charts from eight countries showed that most patients (n = 765 [63.3%]) received no maintenance therapy after HSCT, 219 (18.1%) received FLT3 inhibitor maintenance therapy, and 224 (18.5%) received other types of maintenance therapy. No systematic differences were observed in healthcare resource utilization across the three groups. Clinical benefit was observed with FLT3 inhibitor maintenance over no maintenance therapy with relapse-free survival (adjusted hazard ratio [HR] 0.57 [95% CI 0.34-0.94], P < .05). FLT3 inhibitor and other maintenance also demonstrated overall survival benefit over no maintenance (adjusted HR 0.50 [95% CI 0.28-0.89] and 0.46 [95% CI 0.23-0.91], respectively; both P < .05). CONCLUSIONS: Real-world maintenance therapies after HSCT in patients with FLT3-mutated AML were heterogeneous. While overall use of healthcare resources was not significantly increased in patients receiving maintenance therapy versus those who did not, clinical outcomes were improved.

The combination of FLT3 and SYK kinase inhibitors is toxic to leukaemia cells with CBL mutations.

Mutations in the E3 ubiquitin ligase CBL, found in several myeloid neoplasms, lead to decreased ubiquitin ligase activity. In murine systems, these mutations are associated with cytokine-independent proliferation, thought to result from the activation of hematopoietic growth receptors, including FLT3 and KIT. Using cell lines and primary patient cells, we compared the activity of a panel of FLT3 inhibitors currently being used or tested in AML patients and also evaluated the effects of inhibition of the non-receptor tyrosine kinase, SYK. We show that FLT3 inhibitors ranging from promiscuous to highly targeted are potent inhibitors of growth of leukaemia cells expressing mutant CBL in vitro, and we demonstrate in vivo efficacy of midostaurin using mouse models of mutant CBL. Potentiation of effects of targeted FLT3 inhibition by SYK inhibition has been demonstrated in models of mutant FLT3-positive AML and AML characterized by hyperactivated SYK. Here, we show that targeted SYK inhibition similarly enhances the effects of midostaurin and other FLT3 inhibitors against mutant CBL-positive leukaemia. Taken together, our results support the notion that mutant CBL-expressing myeloid leukaemias are highly sensitive to available FLT3 inhibitors and that this effect can be significantly augmented by optimum inhibition of SYK kinase.

Effects of the multi-kinase inhibitor midostaurin in combination with chemotherapy in models of acute myeloid leukaemia.

Recently, several targeted agents have been developed for specific subsets of patients with acute myeloid leukaemia (AML), including midostaurin, the first FDA-approved FLT3 inhibitor for newly diagnosed patients with FLT3 mutations. However, in the initial Phase I/II clinical trials, some patients without FLT3 mutations had transient responses to midostaurin, suggesting that this multi-targeted kinase inhibitor might benefit AML patients more broadly. Here, we demonstrate submicromolar efficacy of midostaurin in vitro and efficacy in vivo against wild-type (wt) FLT3-expressing AML cell lines and primary cells, and we compare its effectiveness with that of other FLT3 inhibitors currently in clinical trials. Midostaurin was found to synergize with standard chemotherapeutic drugs and some targeted agents against AML cells without mutations in FLT3. The mechanism may involve, in part, the unique kinase profile of midostaurin that includes proteins implicated in AML transformation, such as SYK or KIT, or inhibition of ERK pathway or proviability signalling. Our findings support further investigation of midostaurin as a chemosensitizing agent in AML patients without FLT3 mutations.

Inhibition of the deubiquitinase USP10 induces degradation of SYK.

BACKGROUND: There is growing evidence that spleen tyrosine kinase (SYK) is critical for acute myeloid leukaemia (AML) transformation and maintenance of the leukemic clone in AML patients. It has also been found to be over-expressed in AML patients, with activating mutations in foetal liver tyrosine kinase 3 (FLT3), particularly those with internal tandem duplications (FLT3-ITD), where it transactivates FLT3-ITD and confers resistance to treatment with FLT3 tyrosine kinase inhibitors (TKIs). METHODS: We have previously described a pharmacological approach to treating FLT3-ITD-positive AML that relies on proteasome-mediated FLT3 degradation via inhibition of USP10, the deubiquitinating enzyme (DUB) responsible for cleaving ubiquitin from FLT3. RESULTS: Here, we show that USP10 is also a major DUB required for stabilisation of SYK. We further demonstrate that degradation of SYK can be induced by USP10-targeting inhibitors. USP10 inhibition leads to death of cells driven by active SYK or oncogenic FLT3 and potentiates the anti-leukemic effects of FLT3 inhibition in these cells. CONCLUSIONS: We suggest that USP10 inhibition is a novel approach to inhibiting SYK and impeding its role in the pathology of AML, including oncogenic FLT3-positive AML. Also, given the significant transforming role SYK in other tumours, targeting USP10 may have broader applications in cancer.

Repurposing of Kinase Inhibitors for Treatment of COVID-19.

The outbreak of COVID-19, the pandemic disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spurred an intense search for treatments by the scientific community. In the absence of a vaccine, the goal is to target the viral life cycle and alleviate the lung-damaging symptoms of infection, which can be life-threatening. There are numerous protein kinases associated with these processes that can be inhibited by FDA-approved drugs, the repurposing of which presents an alluring option as they have been thoroughly vetted for safety and are more readily available for treatment of patients and testing in clinical trials. Here, we characterize more than 30 approved kinase inhibitors in terms of their antiviral potential, due to their measured potency against key kinases required for viral entry, metabolism, or reproduction. We also highlight inhibitors with potential to reverse pulmonary insufficiency because of their anti-inflammatory activity, cytokine suppression, or antifibrotic activity. Certain agents are projected to be dual-purpose drugs in terms of antiviral activity and alleviation of disease symptoms, however drug combination is also an option for inhibitors with optimal pharmacokinetic properties that allow safe and efficacious co-administration with other drugs, such as antiviral agents, IL-6 blocking agents, or other kinase inhibitors.

Current therapies under investigation for COVID-19: potential COVID-19 treatments.

In response to the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), researchers are expeditiously searching for antiviral treatments able to alleviate the symptoms of infection, which can be life-threatening. Here, we provide a general overview of what is currently known about the structure and characteristic features of SARS-CoV-2, some of which could potentially be exploited for the purposes of antiviral therapy and vaccine development. This minireview also covers selected and noteworthy antiviral agents/supportive therapy out of hundreds of drugs that are being repurposed or tested as potential treatments for COVID-19, the disease caused by SARS-CoV-2.

Comparison of effects of midostaurin, crenolanib, quizartinib, gilteritinib, sorafenib and BLU-285 on oncogenic mutants of KIT, CBL and FLT3 in haematological malignancies.

Mutations in two type-3 receptor tyrosine kinases (RTKs), KIT and FLT3, are common in both acute myeloid leukaemia (AML) and systemic mastocytosis (SM) and lead to hyperactivation of key signalling pathways. A large number of tyrosine kinase inhibitors (TKIs) have been developed that target either FLT3 or KIT and significant clinical benefit has been demonstrated in multiple clinical trials. Given the structural similarity of FLT3 and KIT, it is not surprising that some of these TKIs inhibit both of these receptors. This is typified by midostaurin, which has been approved by the US Food and Drug Administration for mutant FLT3-positive AML and for KIT D816V-positive SM. Here, we compare the in vitro activities of the clinically available FLT3 and KIT inhibitors with those of midostaurin against a panel of cells expressing a variety of oncogenic FLT3 or KIT receptors, including wild-type (wt) FLT3, FLT3-internal tandem duplication (ITD), FLT3 D835Y, the resistance mutant FLT3-ITD+ F691L, KIT D816V, and KIT N822K. We also examined the effects of these inhibitors in vitro and in vivo on cells expressing mutations in c-CBL found in AML that result in hypersensitization of RTKs, such as FLT3 and KIT. The results show a wide spectrum of activity of these various mutations to these clinically available TKIs.

Inhibition of USP10 induces degradation of oncogenic FLT3.

Oncogenic forms of the kinase FLT3 are important therapeutic targets in acute myeloid leukemia (AML); however, clinical responses to small-molecule kinase inhibitors are short-lived as a result of the rapid emergence of resistance due to point mutations or compensatory increases in FLT3 expression. We sought to develop a complementary pharmacological approach whereby proteasome-mediated FLT3 degradation could be promoted by inhibitors of the deubiquitinating enzymes (DUBs) responsible for cleaving ubiquitin from FLT3. Because the relevant DUBs for FLT3 are not known, we assembled a focused library of most reported small-molecule DUB inhibitors and carried out a cellular phenotypic screen to identify compounds that could induce the degradation of oncogenic FLT3. Subsequent target deconvolution efforts allowed us to identify USP10 as the critical DUB required to stabilize FLT3. Targeting of USP10 showed efficacy in preclinical models of mutant-FLT3 AML, including cell lines, primary patient specimens and mouse models of oncogenic-FLT3-driven leukemia.

Treatment patterns and healthcare resource utilization in patients with FLT3-mutated and wild-type acute myeloid leukemia: A medical chart study.

OBJECTIVES: To assess real-world treatment patterns and healthcare resource utilization (HRU) among patients with FLT3-mutated (FLT3mut ) and FLT3-wild-type (FLT3wt ) acute myeloid leukemia (AML). METHODS: Data were abstracted from medical charts of patients with AML from 10 countries. Patients were grouped based on their FLT3 mutation status, age (18-64 or ≥65), and whether they were newly diagnosed (ND) or relapsed/refractory (R/R). RESULTS: Charts of 1027 AML patients were included (183 FLT3mut 18-64 ND; 136 FLT3mut ≥65 ND; 181 FLT3mut R/R; 186 FLT3wt 18-64 ND; 159 FLT3wt ≥65 ND; 182 FLT3wt R/R). Substantial heterogeneity was observed in treatment patterns for AML. Among ND patients 18-64, the most common initial treatment was standard-to-intermediate dose cytarabine-based therapies (43.2% for FLT3mut and 55.9% for FLT3wt ); among ND patients ≥65, the most common initial treatment was hypomethylating agent-based therapies (36.0% and 47.2%). Among R/R patients, the most common initial treatment after R/R was best supportive care only (39.8% and 24.7%). HRU was substantial across cohorts during both event-free and post-event periods. CONCLUSIONS: Treatment patterns of AML were heterogeneous and FLT3mut AML was treated more aggressively than FLT3wt disease. HRU was substantial for all cohorts, particularly after relapse or treatment failure.

Identification of novel therapeutic targets in acute leukemias with NRAS mutations using a pharmacologic approach.

Oncogenic forms of NRAS are frequently associated with hematologic malignancies and other cancers, making them important therapeutic targets. Inhibition of individual downstream effector molecules (eg, RAF kinase) have been complicated by the rapid development of resistance or activation of bypass pathways. For the purpose of identifying novel targets in NRAS-transformed cells, we performed a chemical screen using mutant NRAS transformed Ba/F3 cells to identify compounds with selective cytotoxicity. One of the compounds identified, GNF-7, potently and selectively inhibited NRAS-dependent cells in preclinical models of acute myelogenous leukemia and acute lymphoblastic leukemia. Mechanistic analysis revealed that its effects were mediated in part through combined inhibition of ACK1/AKT and of mitogen-activated protein kinase kinase kinase kinase 2 (germinal center kinase). Similar to genetic synthetic lethal approaches, these results suggest that small molecule screens can be used to identity novel therapeutic targets in cells addicted to RAS oncogenes.

Structure-guided development of covalent TAK1 inhibitors.

TAK1 (transforming growth factor-ß-activated kinase 1) is an essential intracellular mediator of cytokine and growth factor signaling and a potential therapeutic target for the treatment of immune diseases and cancer. Herein we report development of a series of 2,4-disubstituted pyrimidine covalent TAK1 inhibitors that target Cys174, a residue immediately adjacent to the 'DFG-motif' of the kinase activation loop. Co-crystal structures of TAK1 with candidate compounds enabled iterative rounds of structure-based design and biological testing to arrive at optimized compounds. Lead compounds such as 2 and 10 showed greater than 10-fold biochemical selectivity for TAK1 over the closely related kinases MEK1 and ERK1 which possess an equivalently positioned cysteine residue. These compounds are smaller, more easily synthesized, and exhibit a different spectrum of kinase selectivity relative to previously reported macrocyclic natural product TAK1 inhibitors such as 5Z-7-oxozeanol.

Studies of TAK1-centered polypharmacology with novel covalent TAK1 inhibitors.

Targeted polypharmacology provides an efficient method of treating diseases such as cancer with complex, multigenic causes provided that compounds with advantageous activity profiles can be discovered. Novel covalent TAK1 inhibitors were validated in cellular contexts for their ability to inhibit the TAK1 kinase and for their polypharmacology. Several inhibitors phenocopied reported TAK1 inhibitor 5Z-7-oxozaenol with comparable efficacy and complementary kinase selectivity profiles. Compound 5 exhibited the greatest potency in RAS-mutated and wild-type RAS cell lines from various cancer types. A biotinylated derivative of 5, 27, was used to verify TAK1 binding in cells. The newly described inhibitors constitute useful tools for further development of multi-targeting TAK1-centered inhibitors for cancer and other diseases.

NOTCH2 and FLT3 gene mis-splicings are common events in patients with acute myeloid leukemia (AML): new potential targets in AML.

Our previous studies revealed an increase in alternative splicing of multiple RNAs in cells from patients with acute myeloid leukemia (AML) compared with CD34(+) bone marrow cells from normal donors. Aberrantly spliced genes included a number of oncogenes, tumor suppressor genes, and genes involved in regulation of apoptosis, cell cycle, and cell differentiation. Among the most commonly mis-spliced genes (>70% of AML patients) were 2, NOTCH2 and FLT3, that encode myeloid cell surface proteins. The splice variants of NOTCH2 and FLT3 resulted from complete or partial exon skipping and utilization of cryptic splice sites. Longitudinal analyses suggested that NOTCH2 and FLT3 aberrant splicing correlated with disease status. Correlation analyses between splice variants of these genes and clinical features of patients showed an association between NOTCH2-Va splice variant and overall survival of patients. Our results suggest that NOTCH2 and FLT3 mis-splicing is a common characteristic of AML and has the potential to generate transcripts encoding proteins with altered function. Thus, splice variants of these genes might provide disease markers and targets for novel therapeutics.

Second generation inhibitors of BCR-ABL for the treatment of imatinib-resistant chronic myeloid leukaemia.

Imatinib, a small-molecule ABL kinase inhibitor, is a highly effective therapy for early-phase chronic myeloid leukaemia (CML), which has constitutively active ABL kinase activity owing to the expression of the BCR-ABL fusion protein. However, there is a high relapse rate among advanced- and blast-crisis-phase patients owing to the development of mutations in the ABL kinase domain that cause drug resistance. Several second-generation ABL kinase inhibitors have been or are being developed for the treatment of imatinib-resistant CML. Here, we describe the mechanism of action of imatinib in CML, the structural basis of imatinib resistance, and the potential of second-generation BCR-ABL inhibitors to circumvent resistance.

Gene expression profiling analysis of CRTC1-MAML2 fusion oncogene-induced transcriptional program in human mucoepidermoid carcinoma cells.

BACKGROUND: Mucoepidermoid carcinoma (MEC) arises from multiple organs and accounts for the most common types of salivary gland malignancies. Currently, patients with unresectable and metastatic MEC have poor long-term clinical outcomes and no targeted therapies are available. The majority of MEC tumors contain a t(11;19) chromosomal translocation that fuses two genes, CRTC1 and MAML2, to generate the chimeric protein CRTC1-MAML2. CRTC1-MAML2 displays transforming activity in vitro and is required for human MEC cell growth and survival, partially due to its ability to constitutively activate CREB-mediated transcription. Consequently, CRTC1-MAML2 is implicated as a major etiologic molecular event and a therapeutic target for MEC. However, the molecular mechanisms underlying CRTC1-MAML2 oncogenic action in MEC have not yet been systematically analyzed. Elucidation of the CRTC1-MAML2-regulated transcriptional program and its underlying mechanisms will provide important insights into MEC pathogenesis that are essential for the development of targeted therapeutics. METHODS: Transcriptional profiling was performed on human MEC cells with the depletion of endogenous CRTC1-MAML2 fusion or its interacting partner CREB via shRNA-mediated gene knockdown. A subset of target genes was validated via real-time RT-PCR assays. CRTC1-MAML2-perturbed molecular pathways in MEC were identified through pathway analyses. Finally, comparative analysis of CRTC1-MAML2-regulated and CREB-regulated transcriptional profiles was carried out to assess the contribution of CREB in mediating CRTC1-MAML2-induced transcription. RESULTS: A total of 808 differentially expressed genes were identified in human MEC cells after CRTC1-MAML2 knockdown and a subset of known and novel fusion target genes was confirmed by real-time RT-PCR. Pathway Analysis revealed that CRTC1-MAML2-regulated genes were associated with network functions that are important for cell growth, proliferation, survival, migration, and metabolism. Comparison of CRTC1-MAML2-regulated and CREB-regulated transcriptional profiles revealed common and distinct genes regulated by CRTC1-MAML2 and CREB, respectively. CONCLUSION: This study identified a specific CRTC1-MAML2-induced transcriptional program in human MEC cells and demonstrated that CRTC1-MAML2 regulates gene expression in CREB-dependent and independent manners. Our data provide the molecular basis underlying CRTC1-MAML2 oncogenic functions and lay a foundation for further functional investigation of CRTC1-MAML2-induced signaling in MEC initiation and maintenance.

Identification of driver and passenger mutations of FLT3 by high-throughput DNA sequence analysis and functional assessment of candidate alleles.

Mutations in the juxtamembrane and kinase domains of FLT3 are common in AML, but it is not known whether alterations outside these regions contribute to leukemogenesis. We used a high-throughput platform to interrogate the entire FLT3 coding sequence in AML patients without known FLT3 mutations and experimentally tested the consequences of each candidate leukemogenic allele. This approach identified gain-of-function mutations that activated downstream signaling and conferred sensitivity to FLT3 inhibition and alleles that were not associated with kinase activation, including mutations in the catalytic domain. These findings support the concept that acquired mutations in cancer may not contribute to malignant transformation and underscore the importance of functional studies to distinguish "driver" mutations underlying tumorigenesis from biologically neutral "passenger" alterations.

Combination therapy with nilotinib for drug-sensitive and drug-resistant BCR-ABL-positive leukemia and other malignancies.

Despite the clinical efficacy achieved with frontline therapies for BCR-ABL-positive disease, such as imatinib and second-generation ABL inhibitors like nilotinib or dasatinib that were originally designed to override insensitivity to imatinib, drug resistance still remains a challenge, especially for patients with advanced-stage chronic myeloid leukemia or Philadelphia chromosome-positive acute lymphoblastic leukemia. The discovery of BCR-ABL point mutations has been a great asset to furthering our understanding of a major cause of drug resistance, as has discovery of multidrug resistance proteins, dysregulation of signaling molecules downstream of BCR-ABL, and insights into the underlying causes of stromal-mediated chemoresistance. Such elucidation of mechanisms of resistance associated with leukemic cell survival is essential for the optimization of current therapies and enhancement of patient survival via delaying or preventing disease recurrence. Here, we present an overview of the use of nilotinib in combination with other agents against BCR-ABL-positive leukemia, as well as solid tumors, for the purpose of increasing clinical efficacy and overriding drug resistance.

BRD9 regulates normal human hematopoietic stem cell function and lineage differentiation.

Bromodomain containing protein 9 (BRD9), a member of the non-canonical BRG1/BRM-associated factor (ncBAF) chromatin remodeling complex, has been implicated as a synthetic lethal target in AML but its function in normal human hematopoiesis is unknown. In hematopoietic stem and progenitor cells (HSPC) genomic or chemical inhibition of BRD9 led to a proliferative disadvantage and loss of stem cells in vitro. Human HSPCs with reduced BRD9 protein levels produced lower numbers of immature mixed multipotent GEMM colonies in semi-solid media. In lineage-promoting culture conditions, cells with reduced BRD9 levels failed to differentiate into the megakaryocytic lineage and showed delayed differentiation into erythroid cells but enhanced terminal myeloid differentiation. HSPCs with BRD9 knock down (KD) had reduced long-term multilineage engraftment in a xenotransplantation assay. An increased number of downregulated genes in RNAseq analysis after BRD9 KD coupled with a gain in chromatin accessibility at the promoters of several repressive transcription factors (TF) suggest that BRD9 functions in the maintenance of active transcription during HSC differentiation. In particular, the hematopoietic master regulator GATA1 was identified as one of the core TFs regulating the gene networks modulated by BRD9 loss in HSPCs. BRD9 inhibition reduced a GATA1-luciferase reporter signal, further suggesting a role for BRD9 in regulating GATA1 activity. BRD9 is therefore an additional example of epigenetic regulation of human hematopoiesis.