ABL Tyrosine Kinase Inhibitors Research Articles

Pharmacological effects of small molecule BCR-ABL tyrosine kinase inhibitors on platelet function.

Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL fusion protein, such as imatinib (Gleevec), have revolutionized targeted cancer therapies. However, drug resistance and side effects, particularly those affecting hemostasis, continue to pose significant challenges for TKI therapies. As tyrosine kinases serve pivotal roles in platelet hemostatic function, we investigated the potential impact of both established and emerging ABL TKIs on human platelet activities ex vivo Our study included standard-of-care agents (e.g., imatinib and nilotinib), and second-generation ABL inhibitors including ponatinib and bosutinib designed to mitigate drug resistance. Additionally, we explored the effects of allosteric inhibitors targeting the myristoyl pocket of ABL (e.g., asciminib and GNF-2), and novel agents in preclinical development, including ELVN-919, which uniquely exhibits high specificity for the ABL kinase active site. Our findings reveal that while ABL inhibitors such as ponatinib and bosutinib impede platelet activity, highly specific new-generation ABL inhibitors, including first-in-class therapeutics, do not impact platelet function ex vivo Overall, these new insights around the effects of ABL TKIs on platelet function could inform the development of targeted therapies with reduced hematologic toxicities. Significance Statement This study examines the effects of clinically relevant small molecule BCR-ABL tyrosine kinase inhibitors (TKIs) on platelet activity. This analysis includes first-time assessments of agents such as asciminib and ELVN-919 on human platelet function ex vivo, alongside established therapies (e.g., imatinib, ponatinib) with well-characterized effects on platelet function, to discern potential anti-platelet and other effects of BCR-ABL TKIs and inform clinical safety.

Decitabine, venetoclax, and ponatinib for advanced phase chronic myeloid leukaemia and Philadelphia chromosome-positive acute myeloid leukaemia: a single-arm, single-centre phase 2 trial.

Advanced phase Philadelphia chromosome-positive myeloid disease-consisting of chronic myeloid leukaemia in the myeloid blast phase and in the accelerated phase, and Philadelphia chromosome-positive acute myeloid leukaemia-is associated with poor outcomes. Although previous studies have suggested the benefit of chemotherapy and BCR::ABL1 tyrosine kinase inhibitor combinations, the optimal regimen is uncertain and prospective studies for this rare group of diseases are scant. Preclinical and retrospective clinical data suggest possible synergy between the BCL-2 inhibitor venetoclax and BCR::ABL1 tyrosine kinase inhibitors. We therefore aimed to design a study to evaluate the safety and activity of a novel combination of decitabine, venetoclax, and the third-generation BCR::ABL1 tyrosine kinase inhibitor ponatinib in advanced phase Philadelphia chromosome-positive myeloid diseases. For this phase 2 study, patients aged 18 years or older with previously untreated or relapsed or refractory myeloid chronic myeloid leukaemia-blast phase, chronic myeloid leukaemia-accelerated phase, or advanced phase Philadelphia chromosome-positive acute myeloid leukaemia, and an Eastern Cooperative Oncology Group performance status of 0-3 were eligible. Patients were eligible regardless of the number of previous lines of therapy received or previous receipt of ponatinib. Cycle 1 (induction) consisted of a 7-day lead-in of ponatinib 45 mg orally daily (days 1-7), followed by combination therapy with decitabine 20 mg/m2 intravenously on days 8-12, venetoclax orally daily with ramp-up to a maximum dose of 400 mg on days 8-28, and ponatinib 45 mg orally daily on days 8-28. Cycles 2-24 consisted of decitabine 20 mg/m2 intravenously on days 1-5, venetoclax orally 400 mg on days 1-21, and ponatinib orally daily on days 1-28. Response-based dosing of ponatinib was implemented in consolidation cycles, with reduction to 30 mg daily in patients who reached complete remission or complete remission with an incomplete haematological recovery and a reduction to 15 mg daily in patients with undetectable BCR::ABL1 transcripts. The primary endpoint was the composite rate of complete remission or complete remission with incomplete haematological recovery in the intention-to-treat population. Safety was assessed in the intention-to-treat population. This trial was registered with ClinicalTrials.gov (NCT04188405) and is still ongoing. Between July 12, 2020, and July 8, 2023, 20 patients were treated (14 with chronic myeloid leukaemia-blast phase, four with chronic myeloid leukaemia-accelerated phase, and two with advanced phase Philadelphia chromosome-positive acute myeloid leukaemia). The median age was 43 years (IQR 32-58); 13 (65%) patients were male and seven (35%) were female; and 12 (60%) were White, three (15%) were Hispanic, four (20%) were Black, and one (5%) was Asian. 12 (60%) patients had received 2 or more previous BCR::ABL1 tyrosine kinase inhibitors, and 14 (70%) patients had at least one high-risk additional chromosomal abnormality or complex karyotype. The median duration of follow-up was 21·2 months (IQR 14·1-24·2). The complete remission or complete remission with an incomplete haematological recovery rate was 50% (10 of 20 patients); complete remission in one [5%] patient and complete remission with incomplete haematological recovery in nine [45%]). An additional six (30%) patients had a morphologic leukaemia-free state. The most common grade 3-4 non-haematological adverse events were febrile neutropenia in eight (40%) patients, infection in six (30%), and alanine or aspartate transaminase elevation in five (25%). Eight (40%) patients had at least one cardiovascular event of any grade. There were three on-study deaths, none of which was considered related to the study treatment and all from infections in the setting of refractory leukaemia. The combination of decitabine, venetoclax, and ponatinib is safe and shows promising activity in patients with advanced phase chronic myeloid leukaemia, including those with multiple previous therapies or high-risk disease features. Further studies evaluating chemotherapy and venetoclax-based combination strategies using newer-generation BCR::ABL1 tyrosine kinase inhibitors are warranted. Takeda Oncology, the National Institutes of Health, and the National Cancer Institute Cancer Center.

BCR::ABL1 Proteolysis-targeting chimeras (PROTACs): The new frontier in the treatment of Ph+ leukemias?

BCR::ABL1 tyrosine kinase inhibitors (TKIs) have turned chronic myeloid leukemia (CML) from a lethal condition into a chronic ailment. With optimal management, the survival of CML patients diagnosed in the chronic phase is approaching that of age-matched controls. However, only one-third of patients can discontinue TKIs and enter a state of functional cure termed treatment-free remission (TFR), while the remainder require life-long TKI therapy to avoid the recurrence of active leukemia. Approximately 10% of patients exhibit primary or acquired TKI resistance and eventually progress to the blast phase. It is thought that recurrence after attempted TFR originates from CML stem cells (LSCs) surviving despite continued suppression of BCR::ABL1 kinase. Although kinase activity is indispensable for induction of overt CML, kinase-independent scaffold functions of BCR::ABL1 are known to contribute to leukemogenesis, raising the intriguing but as yet hypothetical possibility, that degradation of BCR::ABL1 protein may accomplish what TKIs fail to achieve - eliminate residual LSCs to turn functional into real cures. The advent of BCR::ABL1 proteolysis targeting chimeras (PROTACs), heterobifunctional molecules linking a TKI-based warhead to an E3 ligase recruiter, has moved clinical protein degradation into the realm of the possible. Here we examine the molecular rationale as well as pros and cons of degrading BCR::ABL1 protein. We review reported BCR::ABL1 PROTACs, point out limitations of available data and compoundsand suggest directions for future research. Ultimately, clinical testing of a potent and specific BCR::ABL1 degrader will be required to determine the efficacy and tolerability of this approach.

Gallic Acid Enhances the Efficacy of BCR::ABL1 Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia through Inhibition of Mitochondrial Respiration and Modulation of Oncogenic Signaling Pathways.

While BCR::ABL1 tyrosine kinase inhibitors have transformed the treatment paradigm for chronic myeloid leukemia (CML), disease progression and treatment resistance due to BCR::ABL1-dependent and BCR::ABL1-independent mechanisms remain a therapeutic challenge. Natural compounds derived from plants have significantly contributed to cancer pharmacotherapy. This study investigated the efficacy of an active component of Leea indica, a local medicinal plant, in CML. Using high-performance liquid chromatography-electrospray ionization-mass spectrometry, a chemical constituent from L. indica extract was isolated and identified as gallic acid. Commercially obtained gallic acid was used as a chemical standard. Gallic acid from L. indica inhibited proliferation and induced apoptosis in CML cell lines, as did the chemical standard. Furthermore, gallic acid induced apoptosis and decreased the colony formation of primary CML CD34+ cells. The combination of isolated gallic acid or its chemical standard with BCR::ABL1 tyrosine kinase inhibitors resulted in a significantly greater inhibition of colony formation and cell growth compared to a single drug alone. Mechanistically, CML cells treated with gallic acid exhibited the disruption of multiple oncogenic pathways including ERK/MAPK, FLT3 and JAK/STAT, as well as impaired mitochondrial respiration. Rescue studies showed that gallic acid is significantly less effective in inducing apoptosis in mitochondrial respiration-deficient ρ0 cells compared to wildtype cells, suggesting that the action of gallic acid is largely through the inhibition of mitochondrial respiration. Our findings highlight the therapeutic potential of L. indica in CML and suggest that gallic acid may be a promising lead chemical constituent for further development for CML treatment.

The BCR::ABL1 tyrosine kinase inhibitors ponatinib and nilotinib differentially affect endothelial angiogenesis and signalling.

BCR::ABL1 inhibitors, the treatment of choice for the majority of patients with chronic myeloid leukaemia (CML), can cause vascular side effects that vary between agents. The exact underlying mechanisms are still poorly understood, but the vascular endothelium has been proposed as a site of origin. The present study investigates the effects of three BCR::ABL1 inhibitors, ponatinib, nilotinib and imatinib, on angiogenesis and signalling in human endothelial cells in response to vascular endothelial growth factor (VEGF). The experiments were performed in endothelial cells isolated from human umbilical veins. After exposure to imatinib, ponatinib and nilotinib, the angiogenic capacity of endothelial cells was assessed in spheroid assays. VEGF-induced signalling pathways were examined in Western blotting experiments using different specific antibodies. RNAi technology was used to downregulate proteins of interest. Intracellular cGMP levels were measured by ELISA. Imatinib had no effect on endothelial function. Ponatinib inhibited VEGF-induced sprouting, while nilotinib increased spontaneous and VEGF-stimulated angiogenesis. These effects did not involve wild-type ABL1 or ABL2, as siRNA-mediated knockdown of these kinases did not affect angiogenesis and VEGF signalling. Consistent with their effects on sprouting, ponatinib and nilotinib affected angiogenic pathways in opposite directions. While ponatinib inhibited VEGF-induced signalling and cGMP formation, nilotinib activated angiogenic signalling, in particular phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2). The latter occurred in an epidermal growth factor receptor (EGFR)-dependent manner possibly via suppressing Fyn-related kinase (FRK), a negative regulator of EGFR signalling. Both, pharmacological inhibition of Erk1/2 or EGFR suppressed nilotinib-induced angiogenic sprouting. These results support the notion that the vascular endothelium is a site of action of BCR::ABL1 inhibitors from which side effects may arise, and that the different vascular toxicity profiles of BCR::ABL1 inhibitors may be due to their different actions at the molecular level. In addition, the as yet unknown pro-angiogenic effect of nilotinib should be considered in the treatment of patients with comorbidities associated with pathological angiogenesis, such as ocular disease, arthritis or obesity.

A Higher Neutrophil Count Is Associated with Favorable Achievement of Treatment-Free Remission in Patients with Chronic Myeloid Leukemia Who Received Second Generation Tyrosine Kinase Inhibitor as Frontline Treatment.

ABL1 tyrosine kinase inhibitor discontinuation securely became among the therapeutic goal for chronic myeloid leukemia chronic phase patients (CML-CP). To establish successful prognostic factors for treatment-free remission (TFR), it is necessary to diagnose the patients with high-risk molecular relapse, however, a biomarker for the achievement of TFR has not been completely elucidated. Recent investigations have determined that neutrophils function crucially in cancer immunology. The research was a multicenter retrospective observational study to examine the correlation between TFR and neutrophil counts before TKI discontinuation. The investigation included patients having Philadelphia chromosome-positive CML-CP who attempted the discontinuation of TKIs after a durable deep molecular response between January 2012 and July 2021 at four institutions in Japan. 118 CML-CP patients in total discontinued TKIs and an estimated 36-month TFR rate was 65.1%. 52 patients received second-generation TKIs as frontline. Higher neutrophil count (>3210/μL) at TKIs discontinuation was determined as an independent prognostic variable for TFR in patients who received second-generation TKIs as frontline [(HR, 0.235 (95%, confidence interval (CI) 0.078-0.711); p = 0.010]. The neutrophil-mediated immunomodulation can be a significant component for the effective achievement of TFR in CML supported by our clinical observation.

Disparities in frontline treatment and overall survival in the era of targeted tyrosine kinase inhibitor therapy for chronic myeloid leukemia: 2004-2021.

11187 Background: The treatment of chronic myeloid leukemia (CML) was revolutionized in 2001 with the introduction of the BCR::ABL tyrosine kinase inhibitor (TKI), imatinib. In 2006, dasatinib was approved, followed by nilotinib in 2007. Two other TKIs, bosutinib & ponatinib were approved in 2012, followed by the allosteric ABL myristoyl inhibitor asciminib in 2021, providing multiple options for patients (pts) who have refractory disease. In this study we examine how survival has improved in CML pts with the availability of multiple TKI’s and identify disadvantaged socioeconomic & demographic groups which continue to have disparate survival. Methods: The National Cancer Database was used to identify CML pts diagnosed (dx) from 2004-2021. Demographic, treatment, & overall survival (OS) were compared by era of TKI availability, with pts dx from 2004-2005 considered to be treated in the imatinib era, 2006-2011 for dasatinib & nilotinib, 2012-2020 with access to bosutinib & ponatinib, and 2021 for asciminib. Results: Of 44,993 CML pts identified, 55.8% were male & 82.5% were White, with median age of 58 years. The portion of pts who were untreated (due to contraindication or early death) decreased by each time period, from 17.8% in the imatinib era to only 7.8% in 2021. The rate of upfront transplant decreased from 2.5% from 2004-2005, to 0.8% in 2021. For dx 2004-2005 median OS was 11.5 yrs (95% CI 10.6-12.5). This increased to 13.6 yrs (CI 13.1-14.1), with age-adjusted hazard ratio (HR) of 0.86 (CI 0.81-0.91) for pts dx 2006-2011, with median OS unreached for 2012-2020, HR 0.67 (CI 0.63-0.71, p<0.001); referenced to 2004-2006. Survival at 1-, 5- & 10-yrs was improved for each subsequent time period of dx, with 5-yr OS of 65%, 71%, & 76%, respectively, p<0.001. On multivariate cox regression adjusted for yr of dx, features associated with reduced OS included age (HR 1.06 [95% CI 1.05-1.06], p<0.001 for each yr), Black race (HR 1.11 [95% CI 1.05-1.18], p<0.001), increased comorbidity index (HR 1.94 [95% CI 1.84-2.04], p<0.001 for index ≥2), uninsured (HR 2.20 [95% CI 2.01-2.41], p<0.001), or insured through Medicaid (HR 2.40 [95% CI 2.23-2.58], p<0.001). Conclusions: Survival for CML pts has significantly improved during the last 20 yrs with the availability of each additional TKI, likely related to increased treatment options for pts with resistance or intolerance. Additional TKI options, particularly approval of ponatinib for patients with T315I mutation, have a secondary benefit of decreasing the utilization of allogenic transplant. Though survival has improved for all pts, pts from traditionally underserved populations, including pts who are underinsured or from racial minority groups, have reduced OS. Continued advances in treatment & efforts to improve access to care for underserved populations is vital to achieve equitable survival outcomes in CML.

Results of ponatinib as frontline therapy for chronic myeloid leukemia in chronic phase.

Ponatinib is a third-generation BCR::ABL1 tyrosine kinase inhibitor (TKI) with robust activity in Philadelphia chromosome-positive leukemias. Herein, we report the long-term follow-up of the phase 2 trial of ponatinib in chronic myeloid leukemia in chronic phase. Patients received ponatinib 30 to 45 mg/day. The primary end point was the rate of 6-month complete cytogenetic response (CCyR). The study was held in June 2014 because of the risk of cardiovascular toxicity, requiring patients to change TKI. Fifty-one patients were treated with ponatinib (median dose, 45 mg/day). Median age was 48 years (range, 21-75); 30 (59%) had baseline cardiovascular comorbidities. Median treatment duration was 13 months (range, 2-25). Fourteen patients (28%) discontinued ponatinib because of toxicities, 36 (71%) after the Food and Drug Administration warning/study closure, and one for noncompliance. Dasatinib was the most frequently chosen second-line TKI (n=34; 66%). Among 46 patients evaluable at 6 months, 44 (96%) achieved CCyR, 37 (80%) major molecular response, 28 (61%) MR4, and 21 (46%) MR4.5. The cumulative 6-month rates of CCyR, major molecular response, MR4, and MR4.5 were 96%, 78%, 50%, and 36%, respectively. Durable MR4 ≥24 or ≥60 months was observed in 67% and 51% of patients, respectively. The 24-month event-free survival rate was 97%. After a median follow-up of 128 months, the 10-year overall survival rate was 90%. Eight patients (16%) had serious grade 2 to 3 cardiovascular adverse events, leading to permanent discontinuation in five (10%). Ponatinib yielded high cytogenetic and molecular responses in newly diagnosed chronic myeloid leukemia in chronic phase. Its use in the frontline setting is hindered by arterio-/vaso-occlusive and other severe toxicities.

Outcome of 3q26.2/MECOM rearrangements in chronic myeloid leukemia.

To evaluate the outcomes of patients with 3q26.2/MECOM-rearranged chronic myeloid leukemia (CML). We reviewed consecutive adult patients with 3q26.2/MECOM-rearranged CML between January 1, 1998 and February 16, 2023. Rearrangements of 3q26.2/MECOM were confirmed by conventional cytogenetics, and fluorescence in situ hybridization starting in 2015. We identified 55 patients with MECOM-rearranged CML, including 23 in chronic phase (CP) or accelerated phase (AP) and 32 in blast phase (BP). Nine patients (16%) achieved a major cytogenetic response (MCyR) or deeper. At a median follow-up of 89months, median survival was 14months. The 5-year survival rate was 19% overall, 23% in CML-CP/AP, and 15% in CML-BP. In the 6-month landmark analysis, the 5-year survival rate was 41% for allogeneic stem cell transplantation (allo-SCT) recipients versus 17% for non-recipients (P = 0.050). Multivariate analysis showed that the percentage of marrow blasts and achievement of MCyR or deeper could predict survival. Outcomes of 3q26.2/MECOM-rearranged CML are poor despite the availability of multiple BCR::ABL1 tyrosine kinase inhibitors (TKIs). Third-generation TKIs in combination with novel agents and possible allo-SCT could be considered given the poor outcomes and resistance to second-generation TKIs.

Ponatinib-review of historical development, current status, and future research.

Ponatinib is a third-generation BCR::ABL1 tyrosine kinase inhibitor (TKI) with high potency against Philadelphia chromosome (Ph)-positive leukemias, including T315I-mutated disease, which is resistant to first- and second-generation TKIs. Ponatinib was approved for T315I-mutated chronic myeloid leukemia (CML), CML resistant/intolerant to ≥2 prior TKIs, advanced phase CML and Ph-positive acute lymphoblastic leukemia (ALL) where no other TKIs are indicated, and T315I-mutated CML and Ph-positive ALL. The response-based dosing of ponatinib in chronic phase CML (CP-CML) improved treatment tolerance and reduced the risk of toxicities, including cardiovascular risks. Ponatinib-based therapy also resulted in significantly better outcomes in frontline Ph-positive ALL compared with prior TKIs and is becoming a new standard of care in this setting. As the clinical development of third-generation TKIs and their rational combinations progresses, we envision further transformative changes in the treatment of CML and Ph-positive ALL.

Ponatinib vs Imatinib in Frontline Philadelphia Chromosome–Positive Acute Lymphoblastic Leukemia

In newly diagnosed Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL), disease progression due to acquired resistance to first- or second-generation BCR::ABL1 tyrosine kinase inhibitors is common. Ponatinib inhibits BCR::ABL1 and all single-mutation variants, including T315I. To compare frontline ponatinib vs imatinib in adults with newly diagnosed Ph+ ALL. Global registrational, phase 3, open-label trial in adults aged 18 years or older with newly diagnosed Ph+ ALL. From January 2019 to May 2022, eligible patients at 77 sites were randomized 2:1 to ponatinib (30 mg/d) or imatinib (600 mg/d) with reduced-intensity chemotherapy, followed by single-agent ponatinib or imatinib after the cycle 20 phase of the trial. The last date of follow-up for this analysis was August 12, 2022. Patients received ponatinib, 30 mg/d, or imatinib, 600 mg/d, with reduced-intensity chemotherapy, followed by single-agent ponatinib or imatinib after cycle 20. The ponatinib dose was reduced to 15 mg on achievement of minimal residual disease-(MRD) negative complete remission. The primary end point of this interim analysis was MRD-negative complete remission (≤0.01% BCR::ABL1 [MR4] centrally assessed by reverse transcriptase-quantitative polymerase chain reaction), with complete remission maintained for at least 4 weeks at the end of cycle 3. The key secondary end point was event-free survival. Of 245 patients randomized (median age, 54 years; 133 [54.3%] female), 232 (ponatinib, n = 154; imatinib, n = 78) who had p190 or p210 dominant isoforms verified by the central laboratory were analyzed for the primary end point. The MRD-negative complete remission rate (primary end point) was significantly higher with ponatinib (34.4% [53/154]) vs imatinib (16.7% [13/78]) (risk difference, 0.18 [95% CI, 0.06-0.29]; P = .002). At the data cutoff, event-free survival had not met the prespecified number of events. Median event-free survival was not reached in the ponatinib group and was 29 months in the imatinib group. The most common adverse events were similar between treatment groups. Arterial occlusive events were infrequent and comparable between groups (ponatinib, 2.5%; imatinib, 1.2%). Ponatinib demonstrated a superior rate of MRD-negative complete remission at the end of induction vs imatinib when combined with reduced-intensity chemotherapy in adults with newly diagnosed Ph+ ALL. The safety profile of ponatinib was comparable with imatinib. ClinicalTrials.gov Identifier: NCT03589326.

Combination of an aurora kinase inhibitor and the ABL tyrosine kinase inhibitor asciminib against ABL inhibitor-resistant CML cells

The development of BCR::ABL1-targeting tyrosine kinase inhibitors (TKIs) has improved the prognosis of patients with chronic myeloid leukemia (CML). However, resistance to ABL TKIs can develop in CML patients due to BCR::ABL1 point mutations and CML leukemia stem cell (LSC). Aurora kinases are essential kinases for cell division and regulate mitosis, especially the process of chromosomal segregation. Aurora kinase members also promote cancer cell survival and proliferation. This study analyzed whether aurora kinases were regulated in the progression of CML. It also evaluated the efficacy of the ABL TKI asciminib and the aurora kinase inhibitor LY3295668. The expressions of AURKA and AURKB were higher in the CML cells compared with normal cells using a public database (GSE100026). Asciminib or LY3295668 alone inhibited CML cells after 72 h, and cellular cytotoxicity was increased. The combined use of Asciminib and LY3295668 increased superior efficacy compared with either drug alone. Colony formation was reduced by cotreatment with asciminib and LY3295668. In the cell-cycle analyses, LY3295668 induced G2/M arrest. Cell populations in the sub-G1 phase were observed when cotreating with asciminib and LY3295668. The combination treatment also changed the mitochondrial membrane potential. In addition, AURKA shRNA transfectant cells had increased asciminib sensitivity. Combining asciminib and aurora kinase inhibition enhanced the efficacy and is proposed as a new therapeutic option for patients with CML. These findings have clinical implications for a potential novel therapeutic strategy for CML patients.

Abstract 121: Ponatinib Promotes Endothelial Cell Inflammation And Leukocyte Trafficking Via Tumor Necrosis Factor Signaling: A Potential Mechanism Of Vascular Toxicity

Background: ABL tyrosine kinase inhibitors (TKI) have dramatically improved chronic myeloid leukemia (CML) survival. Due to resistance to the first ABL-TKI imatinib (Ima), most patients are treated with newer agents, including ponatinib (Pon). Although Pon improves cancer remission, the survival benefit is mitigated by a 4-fold increase in arterial thrombosis. The mechanism of Pon-induced arterial thrombosis and the safety of Asciminib (Asc), a newly approved alternative, are not known. CML patients are older and have notably elevated serum TNFa levels. Hypothesis: Since arterial thrombotic events occur when inflamed vascular plaques rupture, we tested the hypothesis that Pon enhances endothelial cell (EC) response to TNFa, promoting EC inflammation and leukocyte trafficking. Methods and Results: In vitro: Human umbilical vein ECs (HUVECs) were treated with Pon, Ima, or Asc at concentrations observed in humans. Adhesion molecules that mediate leukocyte rolling (E- and P-selectin) and adhesion (ICAM1, VCAM1), and TNF receptors (TNFR) 1 and 2 were quantified at 24 hours by qPCR, flow cytometry (FC), and immunoblot. Pon increased TNFR2, P-selectin, VCAM, and ICAM by FC, qPCR, and immunoblot while Ima and Asc had no impact. In primary human coronary artery ECs Pon, but not Asc nor Ima, similarly increased VCAM and P-selectin mRNA. Pretreatment of HUVECs with a nonspecific TNFR inhibitor prevented Pon induction of ICAM1 and P-selectin mRNA. In vivo: Mice were treated with ABL TKIs by oral gavage to achieve drug plasma levels consistent with those in CML patients. After three days, leukocyte trafficking was quantified by intravital microscopy (IVM) in mouse mesenteric vessels. Pon, but not Asc, increased the number of rolling and adherent leukocytes, and increased serum TNFa by ELISA. Treatment with a TNFR inhibitor blocked Pon induced leukocyte trafficking by IVM. Conclusion: Pon, but not Ima nor Asc, induces expression of TNF receptors and adhesion molecules in human ECs in vitro and promotes leukocyte trafficking in vivo. Since inflamed plaques lead to acute arterial thrombosis, this mechanism could contribute to the high risk of arterial thrombosis in Pon-treated cancer patients. If so, Asc may be a safer choice as it does not induce EC inflammation.

Clinical Insights into Structure, Regulation, and Targeting of ABL Kinases in Human Leukemia.

Chronic myeloid leukemia is a multistep, multi-lineage myeloproliferative disease that originates from a translocation event between chromosome 9 and chromosome 22 within the hematopoietic stem cell compartment. The resultant fusion protein BCR::ABL1 is a constitutively active tyrosine kinase that can phosphorylate multiple downstream signaling molecules to promote cellular survival and inhibit apoptosis. Currently, tyrosine kinase inhibitors (TKIs), which impair ABL1 kinase activity by preventing ATP entry, are widely used as a successful therapeutic in CML treatment. However, disease relapses and the emergence of resistant clones have become a critical issue for CML therapeutics. Two main reasons behind the persisting obstacles to treatment are the acquired mutations in the ABL1 kinase domain and the presence of quiescent CML leukemia stem cells (LSCs) in the bone marrow, both of which can confer resistance to TKI therapy. In this article, we systemically review the structural and molecular properties of the critical domains of BCR::ABL1 and how understanding the essential role of BCR::ABL1 kinase activity has provided a solid foundation for the successful development of molecularly targeted therapy in CML. Comparison of responses and resistance to multiple BCR::ABL1 TKIs in clinical studies and current combination treatment strategies are also extensively discussed in this article.

Результаты применения асциминиба, первого аллостерического ингибитора BCR::ABL1-тирозинкиназы, у больных хроническим миелолейкозом со множественной резистентностью к предшествующей терапии

Currently, there is a crucial need for new treatment approaches to overcome the resistance and intolerance of several tyrosine kinase inhibitor (TKI) therapy lines in chronic myeloid leukemia (CML) patients. Asciminib, the first in its class BCR::ABL1-tyrosine kinase inhibitor specifically targeting ABL myristoyl pocket (STAMP), demonstrated efficacy and safety in CML patients with prior TKI therapy failure, including the cases with pan-resistant T315I mutation in the chimeric BCR::ABL1 gene. The present review focuses on the asciminib mechanism of action, the results of both preclinical and clinical phase I and III studies. Due to the favorable cardiovascular toxicity profile of asciminib, the scope of its application can be extended to patients with cardiovascular co-morbidities. Asciminib is registered in the Russian Federation in January 2023, so treatment algorithms for CML patients with ineffectiveness or intolerance of prior therapy should be updated in line with this new option.

Navigating the Management of Chronic Phase CML in the Era of Generic BCR::ABL1 Tyrosine Kinase Inhibitors.

Over the past several years, advances in research, treatment, and market dynamics have impacted treatment strategies in chronic myeloid leukemia in chronic phase (CML-CP). They include the broader availability of cost-effective generic imatinib, and soon other generic second-generation tyrosine kinase inhibitors (TKIs). Access to affordable generics means that all patients with CML-CP should have access to safe and highly effective lifelong therapies. When overall survival is the treatment endpoint, imatinib provides a good treatment value. Second-generation TKIs may be the best frontline strategy when treatment-free remission is the goal. Recent studies have shown maintained efficacy and reduced toxicity when TKIs are used at reduced dosing. Reduced-dose schedules of second-generation TKIs (which are less toxic and induce faster deep molecular responses) may render generic second-generation TKIs a more attractive treatment option. Adjusting the dose of TKI in the presence of mild-to-moderate, or even severe but reversible, adverse events may be preferable to switching to a different TKI. The selection of second-line and beyond therapies depends on the evolving patterns observed with frontline treatment. Dose-adjusted ponatinib schedules have demonstrated improved efficacy and safety in patients resistant to second-generation TKIs or those with T315I-mutated disease. For asciminib, longer-term follow-up is needed to better evaluate its safety and efficacy compared with ponatinib. Allogeneic stem cell transplantation represents a valid alternative to newer-generation TKIs, with a better treatment value when TKIs are priced at >$40,000/year.

Recent findings and advances in treatment of chronic myeloid leukemia

Imatinib, the first ABL-tyrosine kinase inhibitor (TKI), was approved in 2000 for the treatment of chronic myeloid leukemia (CML). Second- and third-generation TKIs, as well as asciminib, which targets a different site of BCR-ABL1 (the myristoyl pocket), were later approved in 2022. Currently, six drugs are approved for the treatment of CML. Revisions to the clinical guidelines for hematopoietic tumors in 2023 provided new guidance on the utility of new agents as well as TKI dose reduction and treatment discontinuation. This article outlines recently reported predictions regarding TKI treatment response, the role of asciminib in the treatment of CML, and development of new agents, as well as the latest findings regarding the current state of TKI treatment discontinuation.

BCR-ABL kinase domain mutations in CML patients, experience from a tertiary care center in North India

BackgroundChronic Myeloid Leukemia is characterized by the presence of the Philadelphia Chromosome (Ph) which contains the BCR::ABL1 fusion gene that occurs due to a reciprocal translocation between chromosomes 9 and 22. This accounts for up to 15 % of all adult leukemias [1]. Most patients treated with first line tyrosine kinase inhibitor (TKI) imatinib achieve durable response but may undergo relapse at some stage [2]. The most important mechanism that may confer imatinib resistance is point mutation within BCR::ABL kinase domain. Other generation ABL tyrosine kinase inhibitors such as dasatinib, nilotinib, bosutinib and ponatinib help to overcome imatinib resistance [3]. Sensitivity of the patient to each of the above TKIs depends upon the individual candidate mutation present. Thus, it is important to perform mutation analysis for effective therapeutic management of CML patients once they show imatinib resistance. We used direct sequencing to identify the different types of mutations responsible for resistance of imatinib treatment from north India. MethodsIn this study, the patient resistance for the imatinib were analyzed for BCR::ABL kinase domain mutation by direct sequencing and the detected mutations along with their percentage prevalence were reported. Results329 patients with CML-CP were analyzed for BCR::ABL kinase domain mutation. Total 66 (20.06 %) patients out of 329 had mutation in at least one of the domains of BCR::ABL conferring resistance to different generations of TKI. Mutations in BCR::ABL kinase domain was observed in different domain of BCR::ABL. ATP binding P-Loop (42.42 %), Direct binding site (36.36 %), C-Loop (10.60 %), A-Loop (6.06 %), SH2 contact (3.03 %), SH3 contact (1.51 %). ConclusionTotal 20.06 % patients (66/329) show mutation in at least one of the structural motifs of BCR-ABL kinase domain, which further confer the resistance to a particular generation of TKI.

Overcoming BCR::ABL1 dependent and independent survival mechanisms in chronic myeloid leukaemia using a multi-kinase targeting approach

BackgroundDespite improved patient outcome using tyrosine kinase inhibitors (TKIs), chronic myeloid leukaemia (CML) patients require life-long treatment due to leukaemic stem cell (LSC) persistence. LSCs reside in the bone marrow (BM) niche, which they modify to their advantage. The BM provides oncogene-independent signals to aid LSC cell survival and quiescence. The bone-morphogenetic pathway (BMP) is one pathway identified to be highly deregulated in CML, with high levels of BMP ligands detected in the BM, accompanied by CML stem and progenitor cells overexpressing BMP type 1 receptors- activin-like kinases (ALKs), especially in TKI resistant patients. Saracatinib (SC), a SRC/ABL1 dual inhibitor, inhibits the growth of CML cells resistant to the TKI imatinib (IM). Recent studies indicate that SC is also a potent ALK inhibitor and BMP antagonist. Here we investigate the efficacy of SC in overcoming CML BCR::ABL1 dependent and independent signals mediated by the BM niche both in 2D and 3D culture.MethodsCML cells (K562 cell line and CML CD34+ primary cells) were treated with single or combination treatments of: IM, SC and the BMP receptors inhibitor dorsomorphin (DOR), with or without BMP4 stimulation in 2D (suspension) and 3D co-culture on HS5 stroma cell line and mesenchymal stem cells in AggreWell and microfluidic devices. Flow cytometry was performed to investigate apoptosis, cell cycle progression and proliferation, alongside colony assays following treatment. Proteins changes were validated by immunoblotting and transcriptional changes by Fluidigm multiplex qPCR.ResultsBy targeting the BMP pathway, using specific inhibitors against ALKs in combination with SRC and ABL TKIs, we show an increase in apoptosis, altered cell cycle regulation, fewer cell divisions, and reduced numbers of CD34+ cells. Impairment of long-term proliferation and differentiation potential after combinatorial treatment also occurred.ConclusionBMP signalling pathway is important for CML cell survival. Targeting SRC, ABL and ALK kinases is more effective than ABL inhibition alone, the combination efficacy importantly being demonstrated in both 2D and 3D cell cultures highlighting the need for combinatorial therapies in contrast to standard of care single agents. Our study provides justification to target multiple kinases in CML to combat LSC persistence.

Highly Sensitive Chip-Based Digital PCR Platform for Quantitative Detection of BCR::ABL1 Transcripts throughout CML Treatment

Introduction: With various BCR::ABL1 tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) has become a manageable disease and current therapeutic goal is treatment-free remission (TFR). To achieve a successful TFR in more patients, a precise assessment of minimal residual disease (MRD) is one of the prerequisite requirements. Since undetectable BCR::ABL1 fusion transcripts by conventional Taqman™-based quantitative polymerase chain reaction (qPCR) is not an indicator for complete eradication of the CML clone, it requires the development of more sensitive technologies. Currently, to overcome some limitations of conventional qPCR, such as rigorous standardization process and variations in sensitivity between laboratories, digital quantitative-PCR (dqPCR) methods have been developed for accurate and precise detection and its use in the routine clinical practice is gradually expanding. However, current dqPCR methods require the inclusion of one or more negative partitions to calculate the concentration using Poisson distribution. Consequently, this leads to a more than a 10-fold decrease in the maximum detectable concentration in a single test compared to conventional qPCR. Therefore, we have developed a novel technology, High Dynamic Range Digital Real-time PCR (so called HDR-drPCR) to achieve wider dynamic range, which allows more stable, accurate and sensitive monitoring of BCR::ABL1 in a timely manner. Methods: For linearity assessment, we used BCR::ABL1 positive (e14a2) K562 cell line diluted to 7 points by 10-fold serial dilutions from 25 to 2.5x10 -5 %IS with BCR::ABL1 negative HL60 cells. In HDR-drPCR, the GUSB gene was employed as a control gene to determine %IS values. For this purpose, the 1 st WHO international genetic reference panel was utilized to calculate the correction factor (CF). With the results from cell lines, we also evaluated equivalence between conventional qPCR and HDR-drPCR using 219 clinical specimens. Among them, 100 samples were determined as DMR (<0.01%IS) based on the qPCR test results In detail, conventional qPCR was performed using Transcriptor First Strand cDNA Synthesis Kit (Roche, USA) for cDNA synthesis and Real-Q BCR-ABL Quantification Kit (Biosewoom, South Korea) for qPCR. For HDR-drPCR (1-step reverse transcriptase PCR), 10 6 to 10 7 copies per reaction of GUSB gene were applied to disposable cartridges of Genotizer™ HDR BCR::ABL1 Major IS detection kit (Optolane Technologies, South Korea) with LOAA platform (Optolane Technologies, Korea). Based on the results, correlation analysis between qPCR and HDR-drPCR was performed using correlation graphs and Blend-Altman plots. Results: Linearity was assessed by the dilution of RNA of K562 with RNA of HL60 using 10-fold serial dilution of up to - 7 log. The result showed accurate linearity (R 2 = 0.9972) at the range of BCR::ABL1/GUSB ratio of 10 1 to 10 -5(%). Notably, HDR-drPCR technology allowed to detect the level of BCR::ABL1 transcript of MR 6.34 (0.00005 %IS) (Figure 1A). The correlation analysis for 209 clinical samples showed that measured values from both qPCR and HDR-drPCR were closely correlated with the R 2 of 0.973 1 (Figure 1B) . The Bland-Altman plot showed a mean bias of 0.159, and the limit of agreement at the 95% confidence interval (LOA95) was calculated as -0.387 to 0.705. Furthermore, the concordance rate between the two methods was 96.17%, as measured by the proportion of samples in which the difference between the two methods was within a factor of 5. This indicates a strong concordance between the two methods. HDR-drPCR reliably detected BCR::ABL1 transcripts in all 90 clinical specimens measured below 0.01%IS (DMR) using qPCR. Additionally, HDR-drPCR detected trace amounts of BCR::ABL1 transcripts in 6 out of 10 specimens that were not detected by qPCR, showcasing its improved sensitivity compared to the conventional qPCR method. Conclusion: Taken together, we believe our high dynamic range real-time digital PCR(HDR-drPCR) may minimize false positive results and maximize the analytical sensitivity of detecting BCR::ABL1 fusion transcripts and thus, would be useful for accurately assessing treatment in routine clinical practice and facilitating the application of treatment-free remission (TFR). We will include these results in further intensive studies focusing on DMR clinical specimens Study support: Funded by Korea Research Foundation