ABL Inhibitors Research Articles

Abstract 161: Regulation of Abl at mitotic centrosomes and survival by ILK in glioblastoma cells

Abstract Many cancer cells possess supernumerary centrosomes, having more than one centrosome at each pole during mitosis which can lead to aberrant mitotic division and cell death. To prevent this fate, cancer cells cluster their centrosomes at two poles during division to divide in a pseudobipolar manner. As the presence of supernumerary centrosomes is rare in normal cells, targeting the declustering of supernumerary centrosomes in cancer cells is a promising strategy for cancer treatment. Integrin-linked kinase (ILK) is an oncogenic protein and chemotherapeutic target shown to be found at the centrosome where it has a role in facilitating centrosome clustering. Inhibition of ILK, via an anti-ILK chemotherapeutic drug QLT-0267, has been shown to increase centrosome declustering causing mitotic arrest and cell death. However, not all cancers cells are susceptible to anti-ILK treatment alone. Therefore, we investigate a combination treatment strategy targeting ILK and another oncogenic kinase, Abelson kinase (Abl). Both ILK and Abl are involved in cancer cell survival, regulated by the same signalling pathways and share common binding partners that occur at the centrosome and are critical for mitosis. The ILK inhibitor QLT-0267 induced a significant sub-G1 peak following propidium iodide FACS analysis and an increase in MTT labelling, indicating that QLT-0267 significantly decreased survival in glioblastoma cell lines. These effects were augmented in the presence of imatinib, an Abl inhibitor. Moreover, QLT-0267 and imatinib, in combination, significantly increased mitotically arrested cells over the ILK inhibitor alone. Increased levels of cytosolic Abl have been associated with its transformative abilities. The ILK inhibitor effects on survival correlated with its ability to decrease cytosolic Abl levels and inhibited Abl’s localization to mitotic centrosomes in dividing glioblastoma cells. These effects were reversed by the proteasomal inhibitor MG132 (a drug that inhibits the degradation of Abl). Together these results indicate that combination treatment with QLT-0267 and imatinib are more effective then QLT-0267 alone at decreasing successful mitoses and survival in glioblastoma cells. Future studies will unravel the relationship between these oncogenic kinases at centrosomes and its effect on cancer cell cycle progression and survival. Citation Format: Jonathan Dresselhuis, Maddisen Brown, Nicole Robinson, Jacqueline Tan, Michael E. Cox, Julia Mills. Regulation of Abl at mitotic centrosomes and survival by ILK in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 161.

An evaluation of asciminib for patients with chronic myeloid leukemia previously treated with ≥2 tyrosine kinase inhibitors

ABSTRACT Introduction To date, five tyrosine kinase inhibitors (TKIs) are available for treating chronic myeloid leukemia (CML) patients in clinical practice. Despite this, a significant proportion of patients will ultimately develop failure to approved TKIs due to intolerance or resistance. Consequently, new treatment approaches are still required in this unmet clinical need. Asciminib, a first-in-class BCR::ABL1 inhibitor Specifically Targeting the ABL Myristoyl Pocket (STAMP), has the potential to overcome resistance/intolerance to prior TKI treatment. Areas covered This review will cover the mechanism of action, pharmacokinetic profile, and clinical data of asciminib based on available information from laboratory studies, clinical trials, and real-world evidence. Expert opinion Recent approval of asciminib will require positioning of this drug in the treatment algorithm of CML patients failing initial TKI therapy. Available data support the lack of cross-intolerance of asciminib with other TKIs and its favorable cardiovascular toxicity profile. In addition, asciminib has demonstrated considerable efficacy in CML patients who have failed at least two TKIs, although preliminary data suggest that this efficacy may be lower in those previously exposed to ponatinib. The introduction of asciminib in clinical practice may represent an important step forward in the management of CML.

Efficacy and safety results from ASCEMBL, a phase 3 study of asciminib versus bosutinib (BOS) in patients (pts) with chronic myeloid leukemia in chronic phase (CML-CP) after ≥2 prior tyrosine kinase inhibitors (TKIs): Week 96 update.

7004 Background: Asciminib is the first BCR::ABL1 inhibitor to specifically target the ABL Myristoyl Pocket (STAMP). In the ASCEMBL primary analysis, asciminib had superior efficacy and better safety/tolerability than BOS in pts with CML-CP after ≥2 prior TKIs. After a median follow-up of 2.3 years (16.5 months’ additional follow-up since primary analysis), we report efficacy and safety results (cutoff: October 6, 2021). Methods: Eligible pts were adults with CML-CP after ≥2 prior TKIs, with intolerance or lack of efficacy per 2013 European LeukemiaNet. Pts were randomized 2:1 to asciminib 40 mg twice daily or BOS 500 mg once daily, stratified by major cytogenetic response (MCyR) status (Ph+ metaphases ≤35%) at baseline. The key secondary endpoint was major molecular response (MMR) rate at wk 96. Results: 233 pts were randomized to asciminib (n=157) or BOS (n=76). At cutoff, treatment was ongoing in 84 (53.5%) and 15 (19.7%) pts, respectively; the most common reason for discontinuation was lack of efficacy in 38 (24.2%) and 27 (35.5%) pts, respectively. MMR rate at wk 96 (per ITT) was higher on asciminib (37.6%) than BOS (15.8%). The difference, adjusting for baseline MCyR, was 21.7% (95% CI, 10.5%-33.0%; 2-sided P=.001). More pts on asciminib than BOS, respectively, had BCR::ABL1IS ≤1% (45.1% vs 19.4%) at wk 96. The probability of maintaining MMR and BCR::ABL1IS ≤1% for ≥72 wk was 96.7% (95% CI, 87.4%-99.2%) and 94.6% (95% CI, 86.2%-97.9%), respectively, on asciminib and 92.9% (95% CI, 59.1%-99.0%) and 95.0% (95% CI, 69.5%-99.3%), respectively, on BOS. Median duration of exposure was 103.1 (range, 0.1-201.1) wk on asciminib and 30.5 (range, 1.0-188.3) wk on BOS. Despite the longer duration of asciminib exposure, safety/tolerability of asciminib continued to be better than that of BOS (Table). No new on-treatment deaths were reported since the primary analysis. Most frequent (>10%) grade ≥3 adverse events (AEs) on asciminib vs BOS were thrombocytopenia (22.4%, 9.2%), neutropenia (18.6%, 14.5%), diarrhea (0%, 10.5%), and increased alanine aminotransferase (0.6%, 14.5%). Conclusions: After >2 years of follow-up, asciminib continued to show superior efficacy and better safety/tolerability vs BOS. Responses were durable, with more pts on asciminib in MMR. Additionally, more pts on asciminib had BCR::ABL1IS ≤1%, a milestone response in later lines associated with improved survival. These results continue to support the use of asciminib as a new CML therapy, with the potential to transform standard of care. Clinical trial information: NCT03106779. [Table: see text]

Imatinib disassembles the regulatory core of Abelson kinase by binding to its ATP site and not by binding to its myristoyl pocket.

It was recently reported (Xie et al., 2022) that the Abelson tyrosine kinase (Abl) ATP-site inhibitor imatinib also binds to Abl's myristoyl binding pocket, which is the target of allosteric Abl inhibitors. This was based on a crystal structure of a truncated Abl kinase domain construct in complex with imatinib bound to the allosteric site as well as further isothermal titration calorimetry (ITC), NMR, and kinase activity data. Although imatinib's affinity for the allosteric site is significantly weaker (10 M) than for the ATP site (10 nM), imatinib binding to the allosteric site may disassemble the regulatory core of Abl, thereby stimulating kinase activity, in particular for Abl mutants with reduced imatinib ATP-site affinity. It was argued that the previously observed imatinib-induced opening of the Abl regulatory core (Skora et al., 2013; Sonti et al., 2018) may be caused by the binding of imatinib to the allosteric site and not to the ATP site. We show here that this is not the case but that indeed imatinib binding to the ATP site induces the opening of the regulatory core at nanomolar concentrations. This agrees with findings that other type-II ATP-site inhibitors (nilotinib, ponatinib) disassemble the regulatory core despite demonstrated negligible binding to the allosteric site.

A new strategy for the rapid identification and validation of direct toxicity targets of psoralen-induced hepatotoxicity

The interaction between small-molecule compounds of traditional Chinese medicine and their direct targets is the molecular initiation event, which is the key factor for toxicity efficacy. Psoralen, an active component of Fructus Psoraleae, is toxic to the liver and has various pharmacological properties. Although the mechanism of psoralen-induced hepatotoxicity has been studied, the direct target of psoralen remains unclear. Thus, the aim of this study was to discover direct targets of psoralen. To this end, we initially used proteomics based on drug affinity responsive target stability (DARTS) technology to identify the direct targets of psoralen. Next, we used surface plasmon resonance (SPR) analysis and verified the affinity effect of the ‘component-target protein’. This method combines molecular docking technology to explore binding sites between small molecules and proteins. SPR and molecular docking confirmed that psoralen and tyrosine-protein kinase ABL1 could be stably combined. Based on the above experimental results, ABL1 is a potential direct target of psoralen-induced hepatotoxicity. Finally, the targets Nrf2 and mTOR, which are closely related to the hepatotoxicity caused by psoralen, were predicted by integrating proteomics and network pharmacology. The direct target ABL1 is located upstream of Nrf2 and mTOR, Nrf2 can influence the expression of mTOR by affecting the level of reactive oxygen species. Immunofluorescence experiments and western blot results showed that psoralen could affect ROS levels and downstream Nrf2 and mTOR protein changes, whereas the ABL1 inhibitor imatinib and ABL1 agonist DPH could enhance or inhibit this effect. In summary, we speculated that when psoralen causes hepatotoxicity, it acts on the direct target ABL1, resulting in a decrease in Nrf2 expression, an increase in ROS levels and a reduction in mTOR expression, which may cause cell death. We developed a new strategy for predicting and validating the direct targets of psoralen. This strategy identified the toxic target, ABL1, and the potential toxic mechanism of psoralen.

Cell‐Active, Reversible, and Irreversible Covalent Inhibitors That Selectively Target the Catalytic Lysine of BCR‐ABL Kinase

AbstractDespite recent interests in developing lysine‐targeting covalent inhibitors, no general approach is available to create such compounds. We report herein a general approach to develop cell‐active covalent inhibitors of protein kinases by targeting the conserved catalytic lysine residue using key SuFEx and salicylaldehyde‐based imine chemistries. We validated the strategy by successfully developing (irreversible and reversible) covalent inhibitors against BCR‐ABL kinase. Our lead compounds showed high levels of selectivity in biochemical assays, exhibited nanomolar potency against endogenous ABL kinase in cellular assays, and were active against most drug‐resistant ABL mutations. Among them, the salicylaldehyde‐containing A5 is the first‐ever reversible covalent ABL inhibitor that possessed time‐dependent ABL inhibition with prolonged residence time and few cellular off‐targets in K562 cells. Bioinformatics further suggested the generality of our strategy against the human kinome.

Cell-Active, Reversible, and Irreversible Covalent Inhibitors That Selectively Target the Catalytic Lysine of BCR-ABL Kinase.

Despite recent interests in developing lysine-targeting covalent inhibitors, no general approach is available to create such compounds. We report herein a general approach to develop cell-active covalent inhibitors of protein kinases by targeting the conserved catalytic lysine residue using key SuFEx and salicylaldehyde-based imine chemistries. We validated the strategy by successfully developing (irreversible and reversible) covalent inhibitors against BCR-ABL kinase. Our lead compounds showed high levels of selectivity in biochemical assays, exhibited nanomolar potency against endogenous ABL kinase in cellular assays, and were active against most drug-resistant ABL mutations. Among them, the salicylaldehyde-containing A5 is the first-ever reversible covalent ABL inhibitor that possessed time-dependent ABL inhibition with prolonged residence time and few cellular off-targets in K562 cells. Bioinformatics further suggested the generality of our strategy against the human kinome.

Protein tyrosine kinase Abl promotes hepatitis C virus particle assembly via interaction with viral substrate activator NS5A

Previously, we reported that knockdown of Abl protein tyrosine kinase by shRNA or pharmacological inhibition suppresses particle assembly of J6/JFH1 strain–derived hepatitis C virus (HCV) in Huh-7.5 cells. However, the detailed mechanism by which Abl regulates HCV replication remained unclear. In this study, we established Abl-deficient (Abl−) cells through genome editing and compared HCV production between Abl− cells expressing WT or kinase-dead Abl and parental Huh-7.5 cells. Our findings revealed that Abl expression was not required from the stages of virus attachment and entry to viral gene expression; however, the kinase activity of Abl was necessary for the assembly of HCV particles. Reconstitution experiments using human embryonic kidney 293T cells revealed that phosphorylation of Tyr412 in the activation loop of Abl was enhanced by coexpression with the viral nonstructural protein 5A (NS5A) and was abrogated by the substitution of NS5A Tyr330 with Phe (Y330F), suggesting that NS5A functions as a substrate activator of Abl. Abl–NS5A association was also attenuated by the Y330F mutation of NS5A or the kinase-dead Abl, and Abl Tyr412 phosphorylation was not enhanced by NS5A bearing a mutation disabling homodimerization, although the association of Abl with NS5A was still observed. Taken together, these results demonstrate that Abl forms a phosphorylation-dependent complex with dimeric NS5A necessary for viral particle assembly, but that Abl is capable of complex formation with monomeric NS5A regardless of tyrosine phosphorylation. Our findings provide the foundation of a molecular basis for a new hepatitis C treatment strategy using Abl inhibitors.

High-throughput ex vivo drug testing identifies potential drugs and drug combinations for NRAS-positive malignant melanoma

Therapy options for patients with metastatic melanoma (MM) have considerably improved over the past decade. However, many patients still need effective therapy after unsuccessful immunotherapy, especially patients with BRAF-negative tumors who lack the option of targeted treatment second line. Therefore, the elucidation of efficient and personalized therapy options for these patients is required. In this study, three patient-derived cancer cells (PDCs) were established from NRAS Q61-positive MM patients. The response of PDCs and five established melanoma cell lines (two NRAS-positive, one wild type, and two BRAF V600-positive) was evaluated toward a panel of 527 oncology drugs using high-throughput drug sensitivity and resistance testing. The PDCs and cell lines displayed strong responses to MAPK inhibitors, as expected. Additionally, the PDCs and cell lines were responsive to PI3K/mTOR, mTOR, and PLK1 inhibitors among other effective drugs currently undergoing clinical trials. Combinations with a MEK inhibitor were tested with other targeted agents to identify effective synergies. MEK inhibitor showed synergy with multikinase inhibitor ponatinib, ABL inhibitor nilotinib, PI3K/mTOR inhibitor pictilisib, and pan-RAF inhibitor LY3009120. The application of the patients’ cancer cells for functional drug testing ex vivo is one step further in the process of identifying potential agents and agent combinations to personalize treatment for patients with MM. Our preliminary study results suggest that this approach has the potential for larger-scale drug testing and personalized treatment applications in our expansion trial. Our results show that drug sensitivity and resistance testing may be implementable in the treatment planning of patients with MM.

Arsenic Trioxide Suppresses Growth of BCR-ABL1 Positive Cells with “Gatekeeper” or Compound Mutation

Introduction:Chronic myeloid leukemia (CML) and 30% of adult acute lymphatic leukemia (ALL) are characterized by the Philadelphia chromosome (Ph +), having a (9;22) chromosomal translocation. The BCR-ABL1 fusion protein is the hallmark of Ph + leukemia. BCR-ABL1 is characterized by constitutively activated ABL1 tyrosine kinase activity that determines its transformation potential. Tyrosine kinase inhibitors (TKI) have greatly improved the overall prognosis of these diseases. However, unsatisfactory responses in advanced disease stages, resistance and long-term tolerability of BCR-ABL1 inhibitors represent major clinical problems. The most important resistance mechanism against TKIs is the acquisition of point mutations within the BCR-ABL1 kinase domain that impair drug binding, restoring the oncoprotein's constitutively active tyrosine kinase activity. The selection of leukemic clones driven by BCR-ABL1 harboring point mutations, such as the E255K, Y253F/H (P-loop), H396R (activation loop) or the T315I (gatekeeper).Second- and third generation TKIs such as nilotinib, dasatinib, and ponatinib effectively overcome point mutation-mediated resistance. Ponatinib is the only U.S. Food and Drug Administration approved TKI with activity against all known BCR-ABL1 point mutations, including BCR-ABL1-T315I. However, the emergence of compound mutations (two mutations within the same BCR-ABL1 allele) has been linked to resistance to all approved TKIs, including ponatinib, posing a clinical challenge with limited treatment options.The anti-cancer agent arsenic trioxide (ATO) has been used to treat patients with acute promyelocytic leukemia (APL). APL patients respond very well to ATO therapy and achieve complete remission, possibly through induction of apoptosis and differentiation. In addition, it has been demonstrated that combined treatment of ATO with interferon or nilotinib significantly suppressed cell proliferation. However, the potential effects of ATO on BCR-ABL1 mutations and especially on compound mutation is not apparent. This study aimed to investigate the role of ATO in BCR-ABL1 resistant mutations, including compound mutation in Ph + leukemias.Methods:We undertook preclinical evaluation of ATO and compared it with approved TKIs e.g. imatinib, nilotinib, dasatinib, ponatinib and ABL inhibitor asciminib, in vitro models of CML and primary patient-derived long term cultures (PD-LTC) of Ph + ALL patients with or without mutation. The effects on mutational resistance were investigated in Ba/F3 cells expressing BCR-ABL1 with T315I mutation and T315I-E255K mutation. For non-mutational resistance, we used PD-LTCs from Ph + ALL patients with different levels of non-mutational drug resistance. Cell proliferation was assessed by XTT.Results:ATO efficiently inhibited the growth of all PD-LTCs in cellular assays at dosages of 200-500nM. It also suppressed the growth of Ph + PD-LTC with non- mutational resistance (BV) and the BCR-ABL1-T315I positive PD-LTC (KO) in this dosage range. In all modelsWe treated Ba/F3 cells expressing native BCR-ABL1, BCR-ABL1-T315I mutation and BCR-ABL1-T315I-E255K (compound mutation) with increasing concentrations of imatinib (250, 500 and 1000nM), nilotinib (100, 200 and 400nM), dasatinib (10, 25 and 50nM), ponatinib (10, 50 and 100nM), asciminib) (ABL allosteric inhibitor) (5, 10 and 20nM) and ATO (0.5, 1.0 and 2.0 µM). We found that all the inhibitors significantly inhibited the proliferation of Ba/F3 cells expressing wild type BCR-ABL1 in a dose-dependent manner. In contrast, the growth of Ba/F3 cells expressing BCR-ABL1-T315I was inhibited by increasing concentration of ponatinib, asciminib and ATO. ATO potently inhibited the most challenging mutation (T315I-E255K) with a clinically relevant concentration (IC50 250nM). All approved ABL kinase inhibitors (AKIs) and allosteric inhibitors like asciminib could not inhibit the growth of Ba/F3 cells expressing BCR-ABL1 compound mutation.Conclusions:Our findings indicate that ATO significantly suppressed the proliferation of cells expressing non-mutated BCR-ABL1, single and compound mutated BCR-ABL1. These results support including ATO in treating patients with Ph + leukemias having BCR-ABL1 resistant single or compound mutati DisclosuresOttmann: Novartis: Honoraria; Amgen: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Fusion: Honoraria; Incyte: Honoraria, Research Funding.

The Impact of T Cell Immunity on Chemotherapy Response in Childhood Acute Lymphoblastic Leukemia

While most childhood acute lymphoblastic leukemia (ALL) can be cured with risk-adapted combination chemotherapy, a significant proportion of patients experience relapse and eventually succumb to leukemia. In fact, ~50% of ALL relapses occur in patients who have excellent early treatment response (i.e., negative minimal residual disease [MRD] at the end of induction therapy), suggesting that long-term remission is determined by factors beyond leukemia cell drug sensitivity. Inherited genetic variants in genes related to host immune functions have been linked to ALL prognosis, pointing to a possible role of immune vigilance against residual leukemia. However, little is known about how and which host immune factors influence the long-term-remission of this cancer.To this end, we first compared leukemia-free survival of murine BCR-ABL1 B-ALL in response to chemotherapy, in immunocompetent mice (C57BL/6J, “B6”) or mice with various T cell deficiencies. Complete loss of all T cells in the host (Tcra KO) led to a drastic increase in leukemia relapse after treatment with ABL inhibitor dasatinib (p=0.0014) or cytotoxic chemotherapy (e.g., dexamethasone [p=0.0002], and mercaptopurine [p=0.0106]). Similar results were observed when we specifically depleted CD4 or CD8 T cells in host mice (p=0.0302 and 0.0010 for CD4 and CD8 depletion, respectively). ABL1 mutation analysis showed that the immunocompetent mice were much more likely to relapse with wild-type BCR-ABL1 B-ALL after dasatinib withdrawal (71.4%). By contrast, most immunocompromised mice (60.0%, 75%, and 80% of TcraKO, CD4 T cell depletion, and CD8 T cell depletion) relapsed with ABL1-T315I B-ALL. Deep sequencing of BCR-ABL1 mutations during dasatinib therapy indicated that drug resistance mutations emerged early in leukemia regardless of immunocompetency of the host, but the lack of T cell immunity strongly predisposed mice to subsequently develop drug resistance and thus relapse. Furthermore, immunocompetent mice that achieved long-term remission also rejected re-engraftment of BCR-ABL1 ALL, suggesting immune memory against leukemia established during dasatinib treatment.To explore the molecular basis of T-cell immunity against ALL, we collected peripheral T cells during dasatinib therapy from mice which were eventually cured vs. those experienced relapse and compared their global gene expression pattern. Through expression array-based profiling of bulk T cells, we noted upregulation of memory/effector T cell signature genes in mice achieving long-term cure (especially in CD8 T cells), along with genes associated with TCR signaling and cytokine response (IL12 and IFNG). Similarly, scRNA-seq profiling also confirmed the association of TCR signaling, IL12 and IFNG signaling activation in T cells with the cure of ALL in mice. Single-cell RNA-seq analyses of T cell subpopulation specifically identified differences in memory-like T cells from relapse mice vs. those cured with the same chemotherapy, with the latter showing more robust activation of TCR and cytokine signalings. We thus hypothesized that IL12 and IFNG response signaling are key determinants of successful immune surveillance against ALL. Indeed, our in vivo experiments confirmed that 1) IfngKO mice relapsed much more quickly during dasatinib treatment with drug resistance BCR-ABL1 mutations than immunocompetent mice (p<0.0001), and 2) the addition of IL12 to dasatinib therapy significantly improved leukemia-free survival in immunocompetent mice with BCR-ABL1 B-ALL (p=0.0028).Finally, we validated these findings by evaluating the immune cell composition of peripheral blood samples collected during remission from 105 children with ALL treated on the St. Jude Total Therapy Study XV clinical trial. Using the gene expression-based deconvolution method, we quantified the frequency of 9 types of immune cells. There was a significant association of higher level of total T cell (CD8, CD4 naïve, CD4 memory, follicular helper, regulatory, and gamma delta T cells) with superior event-free survival (p=0.031), whereas other immune cells had no impact on ALL treatment outcomes. Even after adjusting for ALL molecular subtypes and MRD levels, T cell frequency remained prognostic (p=0.034).In conclusion, our results firmly established the role of T cell immunity in the cure of ALL and also pointed to T cell activation as a means of improving treatment outcomes of ALL. DisclosuresPui: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Data Monitoring Committee.

Evaluation of CML TKI Induced Cardiovascular Toxicity and Development of Potential Rescue Strategies in a Zebrafish Model.

Tyrosine kinase inhibitors (TKIs) to BCR-ABL1 have been successfully used to treat chronic myeloid leukemia (CML), however, multiple TKI-associated adverse events have been reported and become an emerging problem in patients. The mechanisms of TKI-induced toxicity are not fully understood and it remains challenging to predict potential cardiovascular toxicity of a compound. In this study, we established a zebrafish model to evaluate potential in vivo cardiovascular toxicity of TKIs. We treated the endothelium labeled Tg(kdrl:EGFP) transgenic zebrafish embryos with TKIs then performed confocal imaging to evaluate their vascular structure and function. We found that among FDA approved CML TKIs, ponatinib (the only approved TKI that is efficacious to T315I mutation) is the most toxic one. We then evaluated safety profiles of several clinical stage kinase inhibitors that can target T315I and found that HQP1351 treatment leads to vasculopathies similar to those induced by ponatinib while the allosteric ABL inhibitor asciminib does not induce noticeable cardiovascular defects, indicating it could be a promising therapeutic reagent for patients with T315I mutation. We then performed proof-of-principle study to rescue those TKI-induced cardiovascular toxicities and found that, among commonly used anti-hypertensive drugs, angiotensin receptor blockers such as azilsartan and valsartan are able to reduce ponatinib or HQP1351 induced cardiovascular toxicities. Together, this study establishes a zebrafish model that can be useful to evaluate cardiovascular toxicity of TKIs as well as to develop strategies to minimize TKI-induced adverse events.

ABL1 and Cofilin1 promote T-cell acute lymphoblastic leukemia cell migration.

The fusion gene of ABL1 is closely related to tumor proliferation, invasion, and migration. It has been reported recently that ABL1 itself is required for T-cell acute lymphoblastic leukemia (T-ALL) cell migration induced by CXCL12. Further experiments revealed that ABL1 inhibitor Nilotinib inhibited leukemia cell migration induced by CXCL12, indicating the possible application of Nilotinib in T-ALL leukemia treatment. However, the interacting proteins of ABL1 and the specific mechanisms of their involvement in this process need further investigation. In the present study, ABL1 interacting proteins were characterized and their roles in the process of leukemia cell migration induced by CXCL12 were investigated. Co-immunoprecipitation in combination with mass spectrometry analysis identified 333 proteins that interact with ABL1, including Cofilin1. Gene ontology analysis revealed that many of them were enriched in the intracellular organelle or cytoplasm, including nucleic acid binding components, transfectors, or co-transfectors. Kyoto Encyclopedia of Genes and Genomes analysis showed that the top three enriched pathways were translation, glycan biosynthesis, and metabolism, together with human diseases. ABL1 and Cofilin1 were in the same complex. Cofilin1 binds the SH3 domain of ABL1 directly; however, ABL1 is not required for the phosphorylation of Cofilin1. Molecular docking analysis shows that ABL1 interacts with Cofilin1 mainly through hydrogen bonds and ionic interaction between amino acid residues. The mobility of leukemic cells was significantly decreased by Cofilin1 siRNA. These results demonstrate that Cofilin1 is a novel ABL1 binding partner. Furthermore, Cofilin1 participates in the migration of leukemia cells induced by CXCL12. These data indicate that ABL1 and Cofilin1 are possible targets for T-ALL treatment.

Synergy and Antagonism between Allosteric and Active‐Site Inhibitors of Abl Tyrosine Kinase

AbstractAllosteric inhibitors of Abl kinase are being explored in the clinic, often in combination with ATP‐site inhibitors of Abl kinase. However, there are conflicting data on whether both ATP‐competitive inhibitors and myristoyl‐site allosteric inhibitors can simultaneously bind Abl kinase. Here, we determine whether there is synergy or antagonism between ATP‐competitive inhibitors and allosteric inhibitors of Abl. We observe that clinical ATP‐competitive inhibitors are not synergistic with allosteric ABL inhibitors, however, conformation‐selective ATP‐site inhibitors that modulate the global conformation of Abl can afford synergy. We demonstrate that kinase conformation is the key driver to simultaneously bind two compounds to Abl kinase. Finally, we explore the interaction of allosteric and conformation selective ATP‐competitive inhibitors in a series of biochemical and cellular assays.

Synergy and Antagonism between Allosteric and Active-Site Inhibitors of Abl Tyrosine Kinase.

Allosteric inhibitors of Abl kinase are being explored in the clinic, often in combination with ATP-site inhibitors of Abl kinase. However, there are conflicting data on whether both ATP-competitive inhibitors and myristoyl-site allosteric inhibitors can simultaneously bind Abl kinase. Here, we determine whether there is synergy or antagonism between ATP-competitive inhibitors and allosteric inhibitors of Abl. We observe that clinical ATP-competitive inhibitors are not synergistic with allosteric ABL inhibitors, however, conformation-selective ATP-site inhibitors that modulate the global conformation of Abl can afford synergy. We demonstrate that kinase conformation is the key driver to simultaneously bind two compounds to Abl kinase. Finally, we explore the interaction of allosteric and conformation selective ATP-competitive inhibitors in a series of biochemical and cellular assays.

Oncogenic Kinase Cascades Induce Molecular Mechanisms That Protect Leukemic Cell Models from Lethal Effects of De Novo dNTP Synthesis Inhibition.

Simple SummaryLeukemic cells show differential sensitivity to apoptosis induction by the clinically relevant drug hydroxyurea. Since resistance to hydroxyurea can pose a therapeutic problem, we searched for mechanisms that protect such cells from the toxic effects of hydroxyurea. We used proteomics followed by mass spectrometry to accomplish this task and noted a loss of the RAF1 kinase in cells that are killed by hydroxyurea. Pharmacological inhibition of RAF1 and its target BCL-XL show that these proteins suppress apoptosis induction. Furthermore, inhibition of their upstream regulators BCR-ABL1 (in chronic myeloid leukemia cells) and FLT3-ITD (in acute myeloid leukemia cells) plus hydroxyurea produced favorable results. This approach may benefit patients that are not successfully treated with tyrosine kinase inhibitors. Taken together, we provide novel insights into strategies that eliminate chronic and acute myeloid leukemia cells with combinations of clinically established and currently tested pharmaceutical agents.The ribonucleotide reductase inhibitor hydroxyurea suppresses de novo dNTP synthesis and attenuates the hyperproliferation of leukemic blasts. Mechanisms that determine whether cells undergo apoptosis in response to hydroxyurea are ill-defined. We used unbiased proteomics to uncover which pathways control the transition of the hydroxyurea-induced replication stress into an apoptotic program in chronic and acute myeloid leukemia cells. We noted a decrease in the serine/threonine kinase RAF1/c-RAF in cells that undergo apoptosis in response to clinically relevant doses of hydroxyurea. Using the RAF inhibitor LY3009120, we show that RAF activity determines the sensitivity of leukemic cells toward hydroxyurea. We further disclose that pharmacological inhibition of the RAF downstream target BCL-XL with the drug navitoclax and RNAi combine favorably with hydroxyurea against leukemic cells. BCR-ABL1 and hyperactive FLT3 are tyrosine kinases that causally contribute to the development of leukemia and induce RAF1 and BCL-XL. Accordingly, the ABL inhibitor imatinib and the FLT3 inhibitor quizartinib sensitize leukemic cells to pro-apoptotic effects of hydroxyurea. Moreover, hydroxyurea and navitoclax kill leukemic cells with mutant FLT3 that are resistant to quizartinib. These data reveal cellular susceptibility factors toward hydroxyurea and how they can be exploited to eliminate difficult-to-treat leukemic cells with clinically relevant drug combinations.

ABL001 with Catalytic-Site Inhibitors Limit the CML Immature Cell Population: Re-Differentiation of CML-iPSCs Study

Background:There is growing evidence that chronic myeloid leukemia stem cells (CML-SCs) use survival signals distinct from the BCR-ABL1 kinase to maintain their viability in the presence of ABL-tyrosine kinase inhibitors (TKIs). Understanding the mechanisms through which CML-SCs are resistant to ABL TKIs would help in designing future therapies leading to complete eradication. Induced pluripotent stem cells (iPSCs) are promising for studying stem cell signaling pathways. When reprogramming is performed with CML cells, CML-derived iPSCs offer a unique system for modeling CML pathogenesis and developing high throughput drug screening. ABL001(asciminib) is a potent and selective allosteric ABL1 inhibitor that is undergoing clinical development including CML. In the present study, we investigated the molecular mechanisms by which ABL001 and ponatinib regulate the CML-SCs developing from CML-derived iPSCs.Methods:We generated iPSCs from bone marrow mononuclear cells of three CML patients including CML-CP and CML-AP. Karyotyping analysis revealed that CML-derived iPSCs have Philadelphia chromosome (Ph). In vitro re-differentiation of CML-iPSCs was performed with differentiation media (30 ng/ml VEGF, 30 ng/ml BMP-4, 40 ng/ml SCF, 50 ng/ml Activin) for 4 days. At day 14, a single cell suspension expressing CD34+CD38- was achieved with hematopoietic cytokine (300 ng/ml Flt-3 ligand, 10 ng/ml IL-3, 10 ng/ml IL-6, 50 ng/ml G-CSF, 25 ng/ml BMP-4).Results:We generated the phenotypically immature cell population (CD34+CD38-CD90+CD45+) and mature cell population (CD34-CD45+) from CML-derived iPSCs. The single treatment of ABL001 or ponatinib significantly reduced the mature cell population (CD34-CD45+) from CML-derived iPSCs. Although the single treatment of ABL001 or ponatinib alone have minimum effect on immature cell population (CD34+CD38-CD90+CD45+), co-treatment with ABL001 and ponatinib significantly reduce the cell population (CD34+CD38- CD90+CD45+) (p<0.001). Also co-treatment with ABL001 and ponatinib significantly reduced the colony formations of mature erythroid, granulocyte-macrophade, and mixed of these hematopoietic cells derived from CML-iPSCs for CFC assay. Further, NOD/SCID mice were injected with CML-iPSCs, and then treated with ABL001 and ponatinib on day 21 for 7 days. Co-treatments with ABL001 and ponatinib significantly reduced the population of CD34+CD38-CD90+ cells compared with ABL001 or ponatinib as single agents. To identify the mechanisms that limit the CML immature cell population by ABL001 and ponatinib, CD34+CD38-CD90+CD45+ cell fractions from CML-iPSCs were cultured with ABL001 and or ponatinib for 72 hrs. Co-treatment with ABL001 and ponatinib reduced the expression of BMI1, c-Myc, Oct4 and Nanog and induced the expression of p21Cip1. Finally, we investigate the engraftment potential of CML-derived iPSCs treated with ABL001 and ponatinib. One million of CD34+ cells treated with ABL001 and ponatinib were intravenously transplanted into NOD/SCID mouse. Transient engraft was observed from control mice, however, co-treatment of ABL001 and ponatinib never shows the engraftment.Conclusion:Our preclinical results indicate that combination with ABL001 and ponatinib have potential as an important option for controlling the immature CML cell population. It is expected that ABL001 with catalytic-site inhibitors may become extremely useful therapeutic interventions to achieve treatment-free remission in patients with CML. DisclosuresTauchi:Otsuka pharmaceutical KK: Speakers Bureau; Phizer KK: Research Funding, Speakers Bureau; Celegen KK: Speakers Bureau; Bristol-Myers Squibb KK: Speakers Bureau; Novartis pharma KK: Research Funding, Speakers Bureau. Ohyashiki:Celegen KK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Phizer KK: Honoraria, Research Funding, Speakers Bureau; Jansen pharmaceutical KK: Honoraria, Research Funding; Fujirebio KK: Honoraria; Celegen KK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Nippon Shinyaku: Honoraria, Research Funding, Speakers Bureau; Dainippon Sumitomo KK: Honoraria, Research Funding, Speakers Bureau; Fujirebio KK: Honoraria; Kyowa Hakko Kirin KK: Honoraria; Taiho pharmaceutical KK: Honoraria; Alexion: Honoraria; Novartis pharma KK: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb KK: Honoraria, Research Funding, Speakers Bureau; Otsuka pharmaceutical KK: Honoraria; Jansen pharmaceutical KK: Honoraria, Research Funding; Chugai pharmaceutical KK: Research Funding; Otsuka pharmaceutical KK: Honoraria; Alexion: Honoraria; Taiho pharmaceutical KK: Honoraria; Bristol-Myers Squibb KK: Honoraria, Research Funding, Speakers Bureau; Nippon Shinyaku: Honoraria, Research Funding, Speakers Bureau; Dainippon Sumitomo KK: Honoraria, Research Funding, Speakers Bureau; MSD KK: Honoraria; Kyowa Hakko Kirin KK: Honoraria; Chugai pharmaceutical KK: Research Funding; MSD KK: Honoraria; Phizer KK: Honoraria, Research Funding, Speakers Bureau; Novartis pharma KK: Honoraria, Research Funding, Speakers Bureau.

Crizotinib acts as ABL1 inhibitor combining ATP-binding with allosteric inhibition and is active against native BCR-ABL1 and its resistance and compound mutants BCR-ABL1T315I and BCR-ABL1T315I-E255K

Resistance remains the major clinical challenge for the therapy of Philadelphia chromosome–positive (Ph+) leukemia. With the exception of ponatinib, all approved tyrosine kinase inhibitors (TKIs) are unable to inhibit the common “gatekeeper” mutation T315I. Here we investigated the therapeutic potential of crizotinib, a TKI approved for targeting ALK and ROS1 in non-small cell lung cancer patients, which inhibited also the ABL1 kinase in cell-free systems, for the treatment of advanced and therapy-resistant Ph+ leukemia. By inhibiting the BCR-ABL1 kinase, crizotinib efficiently suppressed growth of Ph+ cells without affecting growth of Ph− cells. It was also active in Ph+ patient-derived long-term cultures (PD-LTCs) independently of the responsiveness/resistance to other TKIs. The efficacy of crizotinib was confirmed in vivo in syngeneic mouse models of BCR-ABL1- or BCR-ABL1T315I-driven chronic myeloid leukemia–like disease and in BCR-ABL1-driven acute lymphoblastic leukemia (ALL). Although crizotinib binds to the ATP-binding site, it also allosterically affected the myristol binding pocket, the binding site of GNF2 and asciminib (former ABL001). Therefore, crizotinib has a seemingly unique double mechanism of action, on the ATP-binding site and on the myristoylation binding pocket. These findings strongly suggest the clinical evaluation of crizotinib for the treatment of advanced and therapy-resistant Ph+ leukemia.

GZD824 overcomes FGFR1-V561F/M mutant resistance in vitro and in vivo.

Abnormallyactivated FGFR1 has been validated as a therapeutic target for differentcancers. Although a variety of FGFR inhibitors have shown benefit in manyclinical patients with FGFR1 aberration, FGFR1 mutant resistance such as V561Mmutation, has been reported. To date however, no FGFR inhibitors have beenapproved to treat patients with FGFR mutant resistance. Herein, we report that GZD824, athird generation ABL inhibitor (Phase II, China), overcomes FGFR1‐V561F/M mutant resistance in vitro and in vivo. GZD824potently suppresses FGFR1/2/3 with an IC50 value of 4.14 ± 0.96, 2.77 ± 0.082, and 8.10 ± 0.15 nmol/L. It effectively overcomes FGFR1‐V561F/M and other mutantresistance in Ba/F3 stable cells (IC50:8.1–55.0 nM), and effectively inhibits the growth of Ba/F3‐FGFR1‐V561F/M mutantxenograft tumors in vivo (TGI=73.4%, 49.8% at20mg/kg, p.o, q2d). GZD824may be considered to be an effective drug to treat patients with FGFR1 abnormalactivation or mutant resistance in clinical trials.

The future belongs to those who prepare for it today

Hippocampal neural stem cells (NSCs) and neurogenesis decline sharply with age, though a small population remains. Two articles in this issue of Cell Stem Cell by Ibrayeva etal. (2021) and Harris etal. (2021) indicate the presence of subpopulations of NSCs whose dynamics of activation and self-renewal change over time and may be key to NSC preservation.