Tyrosine Kinase Inhibitors Ponatinib Research Articles

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.

[Artículo traducido] Asciminib para el tratamiento de la leucemia mieloide crónica en tercera línea: análisis coste-efectividad basado en el enfoque de remisión libre de tratamiento

IntroductionThe first targeted therapy in oncology, imatinib, revolutionized chronic myeloid leukemia (CML) treatment and spurred research in targeted therapies for various cancers. CML results from a chromosomal translocation, forming the BCR-ABL1 fusion gene. Asciminib has been recently approved for 3rd-line refractory or intolerant patients. Treatment-free remission (TFR) is attainable with sustained deep molecular response (DMR) and this approach could be incorporated into pharmacoeconomic models. AimsTo establish a cost-effectiveness model comparing asciminib to approved third-generation tyrosine kinase inhibitors (TKIs) (bosutinib and ponatinib) with a focus on achieving TFR. Additionally, the budgetary impact of incorporating asciminib as a therapeutic alternative is assessed. MethodsThis model is based on a Markov chain with seven states. The condition for achieving TFR is to remain for 5 years in DMR state. Efficacy of the model was measured in QALYs, and the costs included in the base case analysis are based in Spain. A probabilistic (PSA) and deterministic analysis (DSA) were carried out to assess the variability of the model. There were achieved two independent models comparing asciminib vs. bosutinib and asciminib vs. ponatinib. ResultsAsciminib, when compared with ponatinib, is a cost-saving alternative, as efficacy is similar between alternatives, and asciminib has a lower cost of 30,275 €. Asciminib showed 4.33 more QALYs and a higher cost (203,591 €) than bosutinib, resulting in an ICER of €47,010.49 per QALY. PSA shows that the parameters with higher influence in the variability of the model were the probability of transitioning to BP and probabilities of achieving MMR and DMR. A one-way analysis reports that the drug cost has a higher influence on both models, and the discount rate significantly affects the asciminib vs. bosutinib model. ConclusionAsciminib broadens therapeutic choices for patient’s refractory or intolerant to two prior lines of treatment in a cost-effective manner. The costs of drugs significantly impact the overall cost of the disease, emphasizing the importance of the selected discount rates for each drug. Given the relatively low incidence of CML, the introduction of asciminib has a limited budgetary impact, warranting individualized decisions based on patient`s clinical characteristics.

Abstract 6295: Role of MAPK11 in the protective mechanisms against toxicity of tyrosine kinase inhibitors

Abstract Introduction and purpose of the study: Targeted therapies with Tyrosine Kinase Inhibitors (TKIs) have drastically improved the survival of cancer patients. However, long term exposure to TKIs has been associated with severe adverse events such as heart failure, impairing patient survival and life quality, and limiting treatment options. Cardioprotective effect of MAPK11 has been described against cardiac ischemic disease. In addition, MAPK11 has also been reported as an important mediator in cancer development and progression. Therefore the aim of this study is to evaluate the potential therapeutic and dual anti-cancer and cardio-protective effect of MAPK11 inhibition during TKI treatment. Experimental procedures: For this study we used the TKI ponatinib as a paradigm of cardiac toxicity. Ponatinib has been used as a gold standard therapy for the drug resistant type of Chronic Myeloid Leukemia (CML) due to T315I-ABL1 mutation, and has been associated with high risk of cardiac toxicity. We performed in vitro functional assays to evaluate the cardiotoxicity and cardiac protection using human iPSC-cardiomyocytes (hiPSC-CMs), and anti-cancer effect in T315I mutated KCL-22 CML cells. The cardiotoxicity was evaluated by impairment of cardiomyocytes contractile function during TKI (ponatinib) treatment, the cardiac-protection was evaluated by restoration of contractile function post-inhibition of MAPK11 with RNA silencing (siRNA) technique. The anti-cancer effect was evaluated using a cell viability and proliferation assay during inhibition of MAPK11 by siRNA and ABL1 inhibition as a positive control. Summary of unpublished data: Pretreatment with siRNA MAPK11 inhibition significantly improved the contractile function of the cardiomyocytes during treatment with ponatinib when compared to those that did not receive MAPK11 inhibition. Moreover, when compared to the positive control of siRNA ABL1 inhibition, the MAPK11 inhibition showed similar anti-cancer effect in the T315I mutated KCL-22 CML cells proliferation and survival assay. Conclusions: Our study suggests that the inhibition of MAPK11 is a potential druggable therapeutic target with dual action of anti-cancer effect for the mutant form of CML, as well as a substantial protective effect against the cardiac toxicity associated with the TKI ponatinib that could bring insights in the development of safer oncological drugs. Citation Format: Anna Pavlovna Hnatiuk, David Staudt, Mark Mercola. Role of MAPK11 in the protective mechanisms against toxicity of tyrosine kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6295.

Combination of Olverembatinib and VP Regimen As First-Line Therapy for Adult Patients with Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

Backgrand ： Ph+ALL is the largest genetically defined subtype in adult ALL and is considered associated with high relapse risk and inferior clinical outcomes. The combination of tyrosine kinase inhibitors (TKI) with intensive chemotherapy has achieved a major step forward in the therapy of Ph+ALL in deep response and long-term survival. The upfront use of third generation TKI ponatinib has improved the frequency of complete molecular response (CMR) and the overall survival rate to a greater extent. More interesting, the data coming from MDACC showed that combination of ponatinib in the chemotherapy in Ph+ALL maybe challenge the need for stem cell transplantation (SCT) in most patients who achieved CMR (defined BCR-ABL1 transcript < 0·01%) within 3 months after induction therapy. But the cardiovascular adverse event(AE) of ponatinib was more and severely frequently occurred in the former reports. Olverembatinib,a new third generation of TKI, which can also suppress the mutation of T315I and showed promising results in the Ph+CML. Herein we designed the clinical trial to invest the efficiency and safety of Olverembatinib combine with VP regimen (OVP) on Ph+ALL patients as the first-line therapy. Motheds: In this multi-centers, phase 2, single-arm trial, adult patients with previously untreated Ph+ALL were sequentially enrolled. The patients had to be aged 18 years or older, have normal cardiac function (defined by ejection fraction above 50%), and have adequate organ function (serum creatinine≦3*ULN，BUN≦3*ULN，ALT≦3*ULN，AST≦3*ULN，serum bilirubin≦3*ULN). They received three cycles of OVP regimen(for 28 days each cycle :olverembatinib 40mg qod d1-28，Vindesine 4mg d1,8,15,22,Prednisone1mg/Kg/d d1-21,0.5mg/kg/dd22-28). Twelve intrathecal injections of cytarabine alternating with methotrexate were designed as central nervous system prophylaxis in the following therapy after the diagnosis. Findings: 32 patients were screened and 29 were eligible from June 21, 2022 until 21 July,2023. 3 Patients were withdrawn from the clinical trial(1 refused therapy after induction,1 early death due to Covid infection, 1 Protocol deviation ).Among all , Male 12 and Female 17. BCR/ABL1 transcript:M-p210 (11),m-p190（18）.The Median Age was 45(19-74) years old. The Median WBC was 24.8(1.06-308.00) *10E9/L. The treatment has resulted in a 100% overall response, including 96% (24/25) CR and 4% (1/25) CRp. As of data collection,23 finished 3cycles of OVP chemotherapy and 82.6% (19/23) got CMR.AS far the rest 6 patients, 1 got CMR after 1 cycle OVP,1 got CMR after 2cycles of OVP, the other 4 patients were undergoing induction therapy. The total CMR rate was 84% (21/25) at any time. The CMR rate was 36%（9/25） ,76%（19/25）and 82.6%(19/23) at 4weeks,8weeks and 12 weeks respectively. Among all the patients finished the 3 cycles, there were 4 patients not reached the CMR and all of them were M-bcr/abl-p210 protein status. The best response was MMR for 3 patients and CR for 1patient. The CMR rate was 70% (7/10) in M-bcr/abl-p210 transcript and 100% (13/13) in that of m-bcr/abl-p190. During a median follow-up of 241 days, one patient died within one month after transplantation, while all the remaining survived without relapse. During induction therapy, 4 patients had platelets consistently above normal values and 4 patients did not experience a further decrease in hemoglobin compared to baseline. The median time for the occurrence of the lowest values of platelets, hemoglobin, and neutrophils was 6th, 12th, and 11th respectively. The most common non-Hematology AE was liver function damage. AE events greater than or equal to grade 3 included 2 cases of elevated Alanine transaminase ,3 cases γ- GT elevation and 2 cases of triglyceride elevation. Other AEs were mild and less than level 3. Discussion: Ongoing efforts exist to optimize therapeutic options in the frontline for Ph+ALL. The future lies in using less cytotoxic and more targeted agents. The first results of our ongoing trial indicate that the OVP regimen is effective in achieving early high rate of CMR in patients with newly diagnosed Ph+ALL as first-line therapy. All these results were achieved with surprisingly few toxic effects.Until now,16 patients underwent ASCT. Will the excellent outcomes be preserved with longer？ Will there be a difference in long-term outcomes between patients who underwent ASCT and those who do not?The trial is ongoing. we need more time to observe.

Empagliflozin restores autophagy and ameliorates ponatinib-induced cardiomyocyte death

Abstract Background The tyrosine kinase inhibitor ponatinib has proven cardiovascular toxicity, for which no counteractive treatment is available at the moment. Sodium-glucose cotransporter type 2 (SGLT-2) inhibitors are approved drugs treat patients with chronic heart failure of different aetiologies. They play direct anticancer activity in SGLT2-expressing cancer cells, independent of glucose metabolism and through inhibition of oxidative phosphorylation. Ongoing studies are now testing empagliflozin (EMPA) in the prevention of left ventricular dysfunction in patients receiving high cumulative doses of anthracyclines. Aims To determine the effect of EMPA on cardiomyocytes in an in vitro model of PON-induced cardiac toxicity, and to assess underlying cardioprotective mechanisms. Methods and Results We exposed cardiomyocytes to PON (1.7 nM) or vehicle (DMSO) in the presence or absence of EMPA (100 and 500 nM) for 24 hours. Compared with vehicle, incubations with PON significantly reduced cell viability at 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, decreased autophagic cells at immunofluorescence assay (1±0.5 vs 6±1 fluorescence intensity/total area, p=0.0006, Figure 1), and increased cell senescence (15±5 vs 4±2 β-gal positivity/tissue area, p<0.02), effects all reverted by EMPA (p<0.01). We observed no significant effects of PON or EMPA on formation of reactive oxygen species. Compared with vehicle, PON significantly reduced serum response factor (SRF)/serum response elements (SRE) binding activity (25±9 vs 218±15 SRE complex abundance, arbitrary units, p<0.001), in parallel with a reduction in cardiac actin expression (0.32±0.06 vs 0.98±0,12 cardiac actin/GAPDH ratio, arbitrary units, p=0.001), which was reversed by EMPA (p=0.001). Conclusion EMPA attenuates PON cardiotoxicity by reducing senescence and enhancing autophagy, apparently independent of antioxidant mechanisms. This study suggests the design of clinical studies aimed at assessing the effects of EMPA on PON-induced cardiotoxicity.Legend to Figure 1.CON, control (vehicle, DMSO); PON, ponatinib; EMPA 100, empagliflozin 100 nM; EMPA 500, empagliflozin 500 nM

Accomplices in Cure: Blinatumomab + Dasatinib Reduces TCR Signaling and Effector Function Stronger Than Ponatinib and Ameliorates T-Cell Exhaustion

The tyrosine kinase inhibitors (TKIs) dasatinib (dasa) and ponatinib (pona) are being tested in clinical trials in philadelphia chromosome-positive (Ph +) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients in combination with the CD19xCD3 bispecific blinatumomab (blina). Pona but not dasa is able to bind the BCR-ABL T315I mutation, which frequently occurs in BCP-ALL patients. Therefore, clinical studies are moving away from dasa and favor pona. Nevertheless, both TKIs in combination with blina have led to striking remission rates in Ph + BCP-ALL (complete response dasa: 98%, Foá 2020; pona: 92%, Jabbour 2023). Interestingly, increased lymphocyte frequencies were reported in patients under blina + dasa therapy (Puzzolo 2021), indicating that the effect of dasa is not limited to toxicity towards ALL cells conferred by BCR-ABL inhibition. Indeed, dasa modulates T-cell function by interfering with TCR signaling (Mestermann 2019). Also, we have shown that resting of T cells by high-dose dasa treatment ameliorates bispecific-induced T-cell exhaustion (Philipp 2022). In contrast, less is known about the effect of pona on T-cell function. Based on these data, we hypothesize that TKIs fine-tune TCR signaling, which ameliorates T-cell exhaustion during continuous blina infusion. Here, we systematically compared the influence of dasa and pona on blina-mediated TCR signaling and T-cell function. Inhibition of T-cell activation and function by dasa or pona at 0.3 - 100 nM was tested in vitro over 3 days: (1) CD69 expression, (2) blina-mediated killing as specific lysis of CD19 + tumor cells (3) T-cell expansion analyzed by dye dilution or CD2+ fold change (FC), (4) cytokine secretion measured by cytokine catch or cytometric bead array. Inhibition of TCR signaling was evaluated using a Jurkat-NFAT T-cell line stimulated for 6h with blina and CD19 + tumor cells. Phosphoflow of pAKT and pZAP70 was performed on CD3/CD28 activated primary human T cells. Toxicity of the TKIs against BCR-ABL +/- cell lines was evaluated after 3 days. T-cell exhaustion upon continuous blina + dasa stimulation was assessed in an in vitro long-term culture system for continuous bispecific exposure (Philipp 2022). Functional assays as described in (1)-(4) were performed every 7 days with isolated T cells from long-term cultures. Both TKIs inhibited T-cell function in a dose-dependent manner. Surprisingly, dasa was more potent than pona at low-intermediate doses in reducing T-cell function, shown by CD69 expression (mean % at TKI = 3 nM: basal = 70, dasa = 25, pona = 64, p<0.0001), cytotoxic function (mean % specific lysis at TKI = 12.5 nM: basal = 55, dasa = 0, pona = 54, p<0.0001), T-cell proliferation (mean % proliferated at TKI = 1.5 nM: basal = 62, dasa = 25, pona = 51, p<0.0001), as well as IFN secretion (normalized mean fluorescence intensity (MFI) at TKI = 12.5 nM: basal = 655, dasa = 0, pona = 924, p<0.0001). We also observed that NFAT engagement was significantly lower in dasa-treated Jurkat-NFAT cells (relative luminescence units at TKI 12.5 nM: basal = 1620, dasa = 392, pona = 996, p<0.001). Similarly, phosphoflow analyses revealed lower levels of pAKT and pZAP70 upon CD3/CD28 + dasa versus pona treatment (MFI of pAKT at TKI = 25 nM: basal = 2347, dasa = 1642, pona = 2045). Importantly, both TKIs induced cell death of BCR-ABL + but not BCR-ABL - cell lines at concentrations >3 nM. Interestingly, 14 days continuously stimulated T cells with blina + dasa (12.5 nM) did not lead to co-expression of inhibitory checkpoints (mean % of PD-1 +Tim-3 +LAG-3 + T cells on day 14: blina = 31, +dasa = 0.6, p<0.01). Furthermore, when isolated from long-term cultures and tested in functional assays, blina + dasa treated T cells maintained high cytokine secretion (mean IL-2 in pg/ml on day 14: blina = 730, blina + dasa = 5638, p<0.01), granzyme B expression (mean MFI ratio of CD8 + on day 14: blina = 96, blina + dasa = 335, p<0.01) and cytotoxic activity (mean % specific lysis on day 14: blina = 61, blina + dasa = 80). Together, these findings indicate that while both TKIs confer comparable toxicity to BCR-ABL + leukemia cells, dasa dampens TCR signaling and T-cell effector function more potently than pona. Strikingly, dasa can ameliorate T-cell exhaustion upon continuous blina exposure. Our data suggest that low-dose TKIs can fine-tune T-cell activity and might play a considerable role for the clinical success of combinatorial bispecific and TKI therapy in Ph + BCP-ALL.

Lyophilization Based Isolation of Exosomes.

Exosomes are nanoscale extracellular vesicles which regulate intercellular communication. They have great potential for application in nanomedicine. However, techniques for their isolation are limited by requirements for advanced instruments and costly reagents. In this study, we developed a lyophilization-based method for isolating exosomes from cultured cells. The isolated exosomes were characterized for protein content using Bradford assay, and for size distribution and shape using scanning electron microscopy (SEM) and nanoparticles tracking analysis (NTA). In addition, CD63, CD9, CD81, HSP70 and TSG101 were evaluated as essential exosomal surface markers using Western blot. Drug loading and release studies were performed to confirm their drug delivery properties using an in vitro model. Exosomes were also loaded with commercial dyes (Cy5, Eosin) for the evaluation of their drug delivery properties. All these characterizations confirmed successful exosome isolation with measurements of less than 150 nm, having a typical shape, and by expressing the known exosome surface protein markers. Finally, tyrosine kinase inhibitors (dasatinib and ponatinib) were loaded on the exosomes to evaluate their anticancer effects on leukemia cells (K562 and engineered Ba/F3-BCR-ABL) using MTT and Annexin-PI assays. The expression of MUC1 protein on the exosomes isolated from MCF-7 cells also indicated that their potential diagnostic properties were intact. In conclusion, we developed a new method for exosome isolation from cultured cells. These exosomes met all the essential requirements in terms of characterization, drug loading and release ability, and inhibition of proliferation and apoptosis induction in Ph+ leukemia cells. Based on these results, we are confident in presenting the lyophilization-based exosome isolation method as an alternative to traditional techniques for exosome isolation from cultured cells.

Signalling inhibition by ponatinib disrupts productive alternative lengthening of telomeres (ALT)

Alternative lengthening of telomeres (ALT) supports telomere maintenance in 10–15% of cancers, thus representing a compelling target for therapy. By performing anti-cancer compound library screen on isogenic cell lines and using extrachromosomal telomeric C-circles, as a bona fide marker of ALT activity, we identify a receptor tyrosine kinase inhibitor ponatinib that deregulates ALT mechanisms, induces telomeric dysfunction, reduced ALT-associated telomere synthesis, and targets, in vivo, ALT-positive cells. Using RNA-sequencing and quantitative phosphoproteomic analyses, combined with C-circle level assessment, we find an ABL1-JNK-JUN signalling circuit to be inhibited by ponatinib and to have a role in suppressing telomeric C-circles. Furthermore, transcriptome and interactome analyses suggest a role of JUN in DNA damage repair. These results are corroborated by synergistic drug interactions between ponatinib and either DNA synthesis or repair inhibitors, such as triciribine. Taken together, we describe here a signalling pathway impacting ALT which can be targeted by a clinically approved drug.

Abstract 3918: Kinase domain duplications of FGFR1 and MET are potential therapeutic targets

Abstract Background: Kinase domain duplication (KDD) is recently recognized as an oncogenic driver. Therapeutic potential has been established in EGFR-KDDs in non-small cell lung cancer, and our group recently showed resistance mechanism in lung cancers harboring EGFR-KDDs treated with EGFR tyrosine kinase inhibitors (TKIs). However, the frequency and oncogenic role of KDDs in other receptor tyrosine kinases has been elusive. Methods: To investigate the frequency of KDDs in advanced cancer patients, we developed in-house algorithm to detect KDDs in high-depth panel sequencing dataset and evaluated its performance using large-scale panel sequencing data from Seoul National University Hospital (SNUH). In short, the algorithm collects reads with soft clips from a modified GATK best practice pipeline for clinical NGS data processing. The collected reads are then grouped according to their matched position and insert size, and evidences of each group (number of splitting and spanning reads) are collected to yield a set of initial candidates. Finally, the initial candidates were manually inspected after quality control by removing false-positive candidates, such as non-target genes or consistent findings. Then, we established cell lines transfected with KDDs of FGFR1 and MET to validate oncogenic role of identified KDDs in the cohort. Using these transformed cell lines, we investigated carcinogenicity with cell viability assay, immunoblot assay and colony formation assay. FGFR TKIs and MET TKIs were used to uncover the possibility of therapeutic targets in the KDDs. Results: Using the in-house algorithm, we identified FGFR1-KDDs from brain tumors (n=4), EGFR-KDDs from lung (n=1) and brain (n=2) tumors, and MET-KDD from a lung tumor (n=1) in a total of 3,932 cancer samples, with 0.2% frequency. All three kinds of KDDs contained full-length of kinase domains of each gene. Ba/F3 cells transfected with the FGFR1-KDD constructs proliferated well without IL-3 (1239- and 1019-fold higer than wild-type FGFR1), while wild-type FGFR1 Ba/F3 cells did not. This growth potential was diminished by FGFR TKIs BGJ398 (IC50, 0.063 ± 0.065nM), PD173074 (IC50, 1.498 ± 1.021nM), and ponatinib (IC50, 5.69 ± 3.05nM). Reduced expressions of downstream pathway proteins including phospho-FGFR1, phospho-Akt, and phospho-Erk were confirmed by immunoblot assays. NIH-3T3 cells transfected with MET-KDD construct also showed growth advantage compared to wild-type MET NIH-3T3 cells. Capmatinib, a MET TKI, inhibited colony formation of MET-KDD NIH-3T3 cells with reduced phospho-MET expression. Conclusion: In this study, we validated newly developed KDD-detection computational methods using targeted DNA sequencing data. Using this algorithm, we found 0.2% of KDDs in 3,932 cancer samples, including EGFR, FGFR1, and MET. In addition to EGFR-KDDs, our study reveals that both FGFR1- and MET-KDD can be oncogenic and therapeutic targets in brain and lung cancer patients. Citation Format: Chaelin Lee, Sungyoung Lee, Hongseok Yun, Jeonghwan Youk, Tae Min Kim, Soyeon Kim, Bhumsuk Keam, Miso Kim, Dong-Wan Kim. Kinase domain duplications of FGFR1 and MET are potential therapeutic targets. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3918.

Abstract 833: Exosomes isolation via lyophilization for Philadelphia-positive leukemia therapy

Abstract Background: Exosomes are extracellular vesicles having less than 200 nm size, secreted extracellularly by almost all types of cells. They regulate intercellular communication by delivering RNAi, proteins, viruses, and soluble factors as inherited payloads. In recent years, the application of exosomes to deliver small molecules have been widely investigated in tissue regeneration, therapeutics, as well as diagnostics. However, the isolation of exosomes has remained challenging mainly due to limitations of specialized instruments and the least cost-effective reagents. In this study, we developed a novel method to isolate exosomes from cultured cells. Experimental: The supernatant from cultured cells was collected and subjected to lyophilization after low-speed centrifugation. The characterization and quantification of exosomes were performed using Bradford assay. Scanning Electron Microscopy (SEM) and Nanoparticles Tracking Analysis (NTA) were carried out for shape and size distribution. Western blot was performed for the evaluation of the exosome specific surface markers (CD63, CD9, CD81, calnexin, HSP70, and TSG101). The as-isolated exosomes were loaded with commercial dyes (Cy5, Eosin) for the evaluation of their drug delivery properties. Furthermore, their drug loading and release were confirmed by loading tyrosine kinase inhibitors (TKIs) as a payload. Finally, the anti-leukemic drugs (dasatinib and ponatinib) were loaded on the isolated exosomes for targeting leukemia cell line i.e., K562 and BCR-ABL expressing Ba/F3 cells in in vitro model, using MTT assay for cell proliferation and Annexin-V/PI for apoptosis detection. Results: Here we found that the isolated exosomes have round shape with an average size of ~130 nm. They expressed known exosome markers including CD63, CD9, CD81, calnexin, HSP70, and TSG101. These exosomes could efficiently target and deliver dyes (Eosin and Cy5) and TKIs (dasatinib and ponatinib) to K562 and Ba/F3-cells expressing BCR-ABL. The TKIs-loaded exosomes inhibited the growth of leukemic cells and induced apoptosis in a dose dependent manner. Conclusion: We claim the development of a new method for active exosome isolation from cell culture. These exosomes could qualify for all the essential characterization, drug loading, and release ability by harnessing proliferation and inducing apoptosis in Ph+ leukemia cells. Citation Format: Fawad Ur Rehman, Rida e Maria Qazi, Zahra Sajid, Afsar Ali Mian. Exosomes isolation via lyophilization for Philadelphia-positive leukemia therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 833.

Matching-adjusted indirect comparison of asciminib versus other treatments in chronic-phase chronic myeloid leukemia after failure of two prior tyrosine kinase inhibitors

PurposeThe current standard of care for chronic-phase chronic myeloid leukemia (CP-CML) is tyrosine kinase inhibitors (TKIs). Treatment recommendations are unclear for CP-CML failing ≥ 2 lines of treatment, partly due to the paucity of head-to-head trials evaluating TKIs. Thus, matching-adjusted indirect comparisons (MAICs) were conducted to compare asciminib with competing TKIs in third- or later line (≥ 3L) CP-CML.MethodsIndividual patient-level data for asciminib (ASCEMBL; follow-up: ≥ 48 weeks) and published aggregate data for comparator TKIs (ponatinib, nilotinib, and dasatinib) informed the analyses. Major molecular response (MMR), complete cytogenetic response (CCyR), and time to treatment discontinuation (TTD) were assessed, where feasible.ResultsAsciminib was associated with statistically significant improvements in MMR by 6 (relative risk [RR]: 1.55; 95% confidence interval [CI]: 1.02, 2.36) and 12 months (RR: 1.48; 95% CI: 1.03, 2.14) vs ponatinib. For CCyR, the results vs ponatinib were similar by 6 (RR: 1.11; 95% CI: 0.81, 1.52) and 12 months (RR: 0.97; 95% CI: 0.73, 1.28). Asciminib was associated with improvements in MMR by 6 months vs dasatinib but with a CI overlapping one (RR 1.52; 95% CI: 0.66, 3.53). Asciminib was associated with statistically significant improvements in CCyR by 6 (RR: 3.57; 95% CI: 1.42, 8.98) and 12 months (RR: 2.03; 95% CI: 1.12, 3.67) vs nilotinib/dasatinib. Median TTD was unreached for asciminib in ASCEMBL. However, post-adjustment asciminib implied prolonged TTD vs nilotinib and dasatinib, but not vs ponatinib.ConclusionThese analyses demonstrate favorable outcomes with asciminib versus competing TKIs, highlighting its therapeutic potential in ≥ 3L CP-CML.

Ponatinib Drives Cardiotoxicity by S100A8/A9-NLRP3-IL-1β Mediated Inflammation.

The tyrosine kinase inhibitor ponatinib is the only treatment option for chronic myelogenous leukemia patients with T315I (gatekeeper) mutation. Pharmacovigilance analysis of Food and Drug Administration and World Health Organization datasets has revealed that ponatinib is the most cardiotoxic agent among all Food and Drug Administration-approved tyrosine kinase inhibitors in a real-world scenario. However, the mechanism of ponatinib-induced cardiotoxicity is unknown. The lack of well-optimized mouse models has hampered the in vivo cardio-oncology studies. Here, we show that cardiovascular comorbidity mouse models evidence a robust cardiac pathological phenotype upon ponatinib treatment. A combination of multiple in vitro and in vivo models was employed to delineate the underlying molecular mechanisms. An unbiased RNA sequencing analysis identified the enrichment of dysregulated inflammatory genes, including a multifold upregulation of alarmins S100A8/A9, as a top hit in ponatinib-treated hearts. Mechanistically, we demonstrate that ponatinib activates the S100A8/A9-TLR4 (Toll-like receptor 4)-NLRP3 (NLR family pyrin domain-containing 3)-IL (interleukin)-1β signaling pathway in cardiac and systemic myeloid cells, in vitro and in vivo, thereby leading to excessive myocardial and systemic inflammation. Excessive inflammation was central to the cardiac pathology because interventions with broad-spectrum immunosuppressive glucocorticoid dexamethasone or specific inhibitors of NLRP3 (CY-09) or S100A9 (paquinimod) nearly abolished the ponatinib-induced cardiac dysfunction. Taken together, these findings uncover a novel mechanism of ponatinib-induced cardiac inflammation leading to cardiac dysfunction. From a translational perspective, our results provide critical preclinical data and rationale for a clinical investigation into immunosuppressive interventions for managing ponatinib-induced cardiotoxicity.

Ph+ ALL in 2022: is there an optimal approach?

Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) carried a very poor prognosis prior to the advent of tyrosine kinase inhibitors (TKIs) that block the activity of the BCR-ABL1 oncoprotein. With improvements in TKI efficacy and allogeneic hematopoietic cell transplantation (HCT), survival has improved over the past 3 decades, and the role of chemotherapy and allogeneic HCT is now changing. Better risk stratification, the application of the third-generation TKI ponatinib, and the use of immunotherapy with the CD19-CD3 bifunctional T-cell engaging antibody blinatumomab in place of chemotherapy has made therapy for Ph+ ALL more tolerable and arguably more efficacious, especially for older patients who comprise most patients with Ph+ ALL.

Capitalizing on paradoxical activation of the mitogen-activated protein kinase pathway for treatment of Imatinib-resistant mast cell leukemia.

Prevention of fatal side effects during cancer therapy of cancer patients with high-dosed pharmacological inhibitors is to date a major challenge. Moreover, the development of drug resistance poses severe problems for the treatment of patients with leukemia or solid tumors. Particularly drug-mediated dimerization of RAF kinases can be the cause of acquired resistance, also called "paradoxical activation." In the present work we re-analyzed the effects of different tyrosine kinase inhibitors (TKIs) on the proliferation, metabolic activity, and survival of the Imatinib-resistant, KIT V560G, D816V-expressing human mast cell (MC) leukemia (MCL) cell line HMC-1.2. We observed that low concentrations of the TKIs Nilotinib and Ponatinib resulted in enhanced proliferation, suggesting paradoxical activation of the MAPK pathway. Indeed, these TKIs caused BRAF-CRAF dimerization, resulting in ERK1/2 activation. The combination of Ponatinib with the MEK inhibitor Trametinib, at nanomolar concentrations, effectively suppressed HMC-1.2 proliferation, metabolic activity, and induced apoptotic cell death. Effectiveness of this drug combination was recapitulated in the human KIT D816V MC line ROSAKIT D816V and in KIT D816V hematopoietic progenitors obtained from patient-derived induced pluripotent stem cells (iPS cells) and systemic mastocytosis patient samples. In conclusion, mutated KIT-driven Imatinib resistance and possible TKI-induced paradoxical activation can be efficiently overcome by a low concentration Ponatinib and Trametinib co-treatment, potentially reducing the negative side effects associated with MCL therapy.

Towards chemotherapy-free treatment of Philadelphia chromosome-positive acute lymphoblastic leukaemia

The addition of tyrosine kinase inhibitors (TKIs) to conventional chemotherapy backbones for adults with newly diagnosed Philadelphia chromosome-positive (Ph-positive) acute lymphoblastic leukaemia has substantially improved treatment outcomes.1,2 However, the long-term outcomes are still unsatisfactory, with treatment-related resistance and toxicity. Over the past 10 years, several clinical trials have incrementally shown improved outcomes and reduced toxicity for these patients. These steps include the upfront use of the third-generation TKI ponatinib, which is active against the gatekeeper mutation, Thr315Ile, in the ABL1 tyrosine kinase domain.

A Phase II Study of the Combination of Decitabine, Venetoclax and Ponatinib in Patients with Chronic Myeloid Leukemia (CML) in Myeloid Blast Phase (MBP) or Philadelphia-Chromosome Positive (Ph+) Acute Myeloid Leukemia (AML)

Background: Advanced phases of CML and Ph+ AML are therapeutic challenges. Preclinical and early clinical data have shown that combining venetoclax (VEN) with a tyrosine kinase inhibitor (TKI) works synergistically in Ph+ myeloid leukemias (Leonard et. al., Sci Transl Med 2016 and Maiti et. al., Acta Haematologica 2020). We hypothesized that combining a potent TKI (ponatinib) with a hypomethylating agent plus VEN backbone might lead to high rates of response that could facilitate potentially curative allogeneic stem cell transplantation (SCT). Methods: This is a single-arm phase II study (NCT04188405) that enrolled patients (pts) ≥ 18 years (yrs) of age with newly diagnosed or relapsed/refractory advanced phase CML [CML in accelerated phase (CML-AP) or CML-MBP] or Ph+ AML. Pts were required to have an ECOG PS ≤ 3 and adequate liver and renal function. Pts with clinically significant cardiovascular comorbidities, including recent or active myocardial infarction, heart failure, clinically significant arrhythmias, cerebrovascular accident, venous thromboembolism or uncontrolled hypertension were excluded. Pts were treated with a combination of decitabine (DAC) (20 mg/m2/day) x 5 days, VEN (400 mg equivalent dose/day) x 21 days, and ponatinib (45mg/day) x 21 days in cycle 1 and then continuously in cycle 2+ (Fig. 1A). Ponatinib was reduced to 30mg/day for pts in CR/CRi and to 15 mg/day for pts in CMR. For C1 only, pts who had not received ponatinib prior to trial initiation had a lead-in phase of ponatinib monotherapy for 7 days before the initiation of DAC and VEN. Cycles were repeated every 28 days, as permitted by peripheral blood count recovery, and pts could receive a maximum of 24 cycles. Intrathecal prophylaxis with 4 doses of cytarabine was recommended. The primary objective was to determine the overall response rate of the regimen. Results: From July 2020 to May 2022, 12 pts were treated, 1 pt with de novo Ph+ AML and 11 pts with advanced phase CML (2 pts with CML-AP and 9 pts with CML-MBP). The median age of the pts was 45 yrs (range, 28-71 yrs). Three pts (25%) had received no prior therapy (1 each with Ph+ AML, CML-AP and CML-MBP). Nine pts (75%) had prior TKI exposure (including prior ponatinib exposure in 4 pts) and 3 pts (25%) had received prior chemotherapy. Eleven pts (92%) had the p210 transcript of BCR::ABL1. Eight pts (67%) had additional cytogenetic abnormalities, including 5 pts with complex karyotype and 2 pts with a MECOM rearrangement. 4 of 11 tested pts (36%) had an ABL1 kinase domain mutation detected at enrollment (T315I, Q252H, L384M and E255K in 1 pt each). The median number of cycles received was 2 (range, 1-7). Nine pts (75%) responded, including 1 with CR, 3 with CRi and 5 with MLFS as best response. The CR/CRi rate was 33%. Best response was achieved after cycle 1 in 7 of 9 responders. Both pts with CML-AP responded, and 2 of 3 pts without prior TKI exposure responded. One of the responders achieved an MMR. After a median follow-up of 9.1 months, the median overall survival (OS) is 9.5 months, and the estimated 9-month OS is 64%. The median relapse-free survival 5.1 months. Among the 9 responders, 3 pts are in continuous response without SCT, 1 pt proceeded to SCT and is still alive, 3 pts did not undergo SCT and relapsed (2 of whom had a complex karyotype), 1 pt died in MLFS, and 1 pt was lost to follow-up (Fig. 1B). The one pt who proceeded to SCT achieved MLFS as best response pre-SCT and remains in MRD-negative CR 22 months post-SCT. The toxicity profile of the regimen was similar to historical expectations with DAC plus VEN. One pt developed grade 3 mucositis, which was considered related to study therapy. No grade 4-5 related non-hematological adverse events were observed. Related grade 1-2 non-hematological adverse events included rash in 2 pts, and transaminitis, creatinine elevation and mucositis in 1 pt each. Of the 8 pts who received > 1 cycle of study therapy, 4 pts had delay (>1 week) in initiation of a subsequent treatment cycle due to cytopenias; for 2 of these pts the duration of VEN was reduced. The 60-day mortality rate was 0%. Two pts died on the study, one from leukemia and one from infection while in MLFS. Conclusion: The triplet combination of DAC, VEN and ponatinib was well-tolerated in pts with advanced Ph+ myeloid leukemias. In this poor-risk population, a marrow remission rate of 75% was achieved, although durations of remission were modest in the absence of consolidative SCT. This study continues to accrue pts. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

BH3 mimetics in combination with nilotinib or ponatinib represent a promising therapeutic strategy in blast phase chronic myeloid leukemia

Dysregulation of the BCL-2 family is implicated in protecting chronic myeloid leukemia (CML) cells from intracellular damage and BCR::ABL1-inhibition with tyrosine kinase inhibitors (TKIs) and may be a viable therapeutic target in blast phase (BP-)CML, for which treatment options are limited. BH3 mimetics, a class of small molecule inhibitors with high-specificity against the prosurvival members of the BCL-2 family, have displayed clinical promise in the treatment of chronic lymphocytic and acute myeloid leukemia as single agents and in combination with standard-of-care therapies. Here we present the first comparison of inhibition of BCL-2 prosurvival proteins BCL-2, BCL-xL and MCL-1 in combination with a second or third generation TKI, crucially with comparisons drawn between myeloid and lymphoid BP-CML samples. Co-treatment of four BP-CML cell lines with the TKIs nilotinib or ponatinib and either BCL-2 (venetoclax), MCL-1 (S63845) or BCL-xL (A-1331852) inhibitors resulted in a synergistic reduction in cell viability and increase in phosphatidylserine (PS) presentation. Nilotinib with BH3 mimetic combinations in myeloid BP-CML patient samples triggered increased induction of apoptosis over nilotinib alone, and a reduction in colony-forming capacity and CD34+ fraction, while this was not the case for lymphoid BP-CML samples tested. While some heterogeneity in apoptotic response was observed between cell lines and BP-CML patient samples, the combination of BCL-xL and BCR::ABL1 inhibition was consistently effective in inducing substantial apoptosis. Further, while BH3 mimetics showed little efficacy as single agents, dual-inhibition of BCL-2 prosurvival proteins dramatically induced apoptosis in all cell lines tested and in myeloid BP-CML patient samples compared to healthy donor samples. Gene expression and protein level analysis suggests a protective upregulation of alternative BCL-2 prosurvival proteins in response to BH3 mimetic single-treatment in BP-CML. Our results suggest that BH3 mimetics represent an interesting avenue for further exploration in myeloid BP-CML, for which alternative treatment options are desperately sought.

Outcomes of Patients with B-ALL with Concomitant BCR::ABL1 and CRLF2 Rearrangements

Background: Outcomes of patients with Philadelphia chromosome positive (Ph+) ALL have improved significantly with the introduction of tyrosine kinase inhibitors (TKI). Ph-like ALL is a high-risk subtype of B-ALL with gene expression profile similar to Ph+ ALL, but without the BCR-ABL translocation. About 50% of the patients with Ph-like ALL have CRLF2 rearrangement (CRLF2-R) and are associated with poor outcomes with conventional chemoimmunotherapy. The co-occurrence of both Ph+ and CRLF2-R is rare (Jain, Haematologica 2017) and long-term outcomes unknown. Methods: We retrospectively analyzed the Leukemia and Pathology databases for patients who presented to the MD Anderson Cancer Center (MDACC) and were noted to have concomitant Ph+ and CRLF2-R. Ph+ was assessed by conventional cytogenetics / FISH / PCR for BCR-ABL1. CRLF2-R was assessed by flow cytometry / FISH. Outcomes assessed were best response (complete remission (CR), relapse, minimal residual disease (MRD), event-free survival (EFS) and overall survival (OS). Results: We identified 8 patients (5 newly diagnosed and 3 R/R B-ALL) who had concurrent Ph+ and CRLF2-R rearrangement (Table 1). The median age was 29 (range, 14-57), 6 (75%) were of Hispanic ethnicity, and 6 (75%) were men. The median clone size of CRLF2-R and Ph+ were 73% (56-92%) and 52% (4-96%), respectively. The most frequent mutated gene was JAK2 in 3/8 (38%) patients. All patients were treated with chemoimmunotherapy or immunotherapy + TKI (ponatinib, n=7; dasatinib, n=1). The median follow-up was 23 months. The best response achieved was CR in 6/8 (75%). The median EFS was 8.6 months; 12-month OS was 75% (Figure 1). A total of 3/6 (50%) responders had a subsequent relapse with 2/3 relapses occurring in the extramedullary compartment (one in central nervous system and one intraperitoneal); both with CRLF2-R clone without Ph+. Of the 5 patients who presented to MDACC as newly diagnosed B-ALL, two were treated with frontline with Hyper-CVAD + ponatinib/dasatinib, two with blinatumomab + ponatinib and the one with a pediatric regimen + dasatinib. Best response was CR with negative MRD in 5/5 (100%). All 5 patients cleared both rearrangements (Ph+ and CRLF2) at the time of remission; 2/5 patients showed CRLF2-R during relapse (without the Ph+). The 12-month OS and EFS were 80% and 60%, respectively. Three patients presented to MDACC with R/R B-ALL. They received HCVAD + ponatinib/dasatinib +/-ruxolitinib. Best response was CR in 1/3 (33%) patients. All 3 patients cleared Ph+, but CRLF2-R persisted in 2 patients. Conclusions: Patients who harbor concomitant Ph+ and CRLF2-R represent a rare but high-risk subgroup of B-ALL. High rate of relapse was noted despite treatment with chemotherapy + TKI +/- ruxolitinib; several patients cleared the Ph+ clone, but CRLF2-R clone persisted suggesting novel interventions against CRLF2-R B-ALL are warranted. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Olverembatinib (HQP1351) Overcomes Ponatinib Resistance in Patients with Heavily Pretreated/Refractory Chronic Myeloid Leukemia (CML) and Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia (Ph+ ALL)

Background Olverembatinib is a novel third-generation BCR-ABL1 tyrosine kinase inhibitor (TKI) with antitumor activity against CML and Ph+ ALL regardless of genotype and a preliminarily favorable human safety profile. This study is the first to report on the safety, efficacy, and pharmacokinetic (PK) profiles of olverembatinib in patients with CML (all phases) and Ph+ ALL outside China, whose disease failed ponatinib treatment and at least 2 prior TKI treatments. Methods In this multicenter, open-label, randomized trial, olverembatinib is administered orally on alternate days (QOD) in adults with chronic-phase (CP-CML), accelerated-phase (AP-CML), or blast-phase CML (BP-CML) or Ph+ ALL. Enrolled patients have had no fewer than 2 prior TKI failures, except for those whose disease harbors T315I mutation, for whom the number of previous TKIs is not limited. Study participants are randomized 3:3:2 to olverembatinib 30, 40, or 50 mg QOD in 28-day cycles. Results A total of 30 patients have been enrolled, including 23 with CP-CML, 4 with AP-CML, 2 with BP-CML, and 1 with Ph+ ALL. The median (range) treatment duration is 4.8 (0.03-21.29) months and the median (range) interval from CML/Ph+ ALL diagnosis to olverembatinib treatment onset, 7.0 (1.5-24.0) years. Half (15/30) of patients are men, and the median (range) age is 47.0 (21.0-74.0) years. In all, 1 (3.3%), 7 (23.3%), 8 (26.7%), and 9 (30.0%) patients received 2, 3, 4, and ≥ 5 prior TKIs, respectively. A total of 21 (70.0%) patients were pretreated with third-generation TKI ponatinib, including 17 (81.0%) with resistance and 4 (19.0%) with intolerance; a total of 5 (16.7%) were pretreated with asciminib; 12 (40.0%) had T315I mutations; and 13 (43.3%), hypertension. At baseline, all patient BCR-ABL1IS levels were > 1%. Olverembatinib was well tolerated. Twenty-two (73.3%) patients experienced any-grade treatment-related AEs (TRAEs; Table 1), incidences of which tended to be dose-dependent. Most nonhematologic TRAEs were grade 1/2. Common grade 3/4 hematologic TRAEs included thrombocytopenia (7/30; 23.3%), neutropenia (5/30; 16.7%), and decreased leukocyte counts (4/30; 13.3%). In all, 11 (36.7%) of 30 patients experienced serious AEs, of which 6 (20.0%) were considered olverembatinib-related and 1 (3.3%) led to treatment discontinuation. One patient with AP-CML from the 50-mg dose cohort died of progressive disease. Olverembatinib conferred potent antileukemic activity in patients with CML and Ph+ ALL (Table 2). Of 21 efficacy-evaluable patients, 17 were evaluable for cytogenetic response, of whom 10 (58.8%) had complete cytogenetic response (CCyR); 9/21 (42.9%) patients had major molecular responses (MMR). Olverembatinib was effective in patients with either T315I-mutant (62.5%, CCyR; 50%, MMR) or T315IWT (55.6%, CCyR; 38.5%, MMR), and its effectiveness was not compromised by prior use of ponatinib or asciminib. Among patients pretreated with ponatinib with available responses, 5 (55.6%) of 9 patients with ponatinib-resistant disease experienced CCyR and 6 (54.5%) of 11, MMR. Four of 5 patients pretreated with asciminib showed response. PK analyses indicated an approximately dose-proportional increase in olverembatinib plasma exposure from 30 to 50 mg QOD and comparable plasma exposure between Chinese and US CML populations. Conclusions Olverembatinib monotherapy is efficacious and well tolerated in patients with TKI-refractory CML and Ph+ ALL. Even in patients with CML who were ponatinib or asciminib resistant, or who had T315I mutations, olverembatinib showed strong efficacy. This analysis confirms that olverembatinib is a potentially viable treatment option for patients with CML and Ph+ ALL, including those with ponatinib failure or T315I mutation. Internal study identifier: HQP1351-CU101. Clinicaltrials.gov identifier: NCT04260022. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 14372: Case Series: Non-Lipid Plaque Formation as the Mechanism for Ponatinib-Induced Vascular Toxicity Using Operator-Assisted Virtual Histology Optical Coherence Tomography

Introduction: The third-generation tyrosine kinase inhibitor ponatinib approved for treating chronic myeloid leukemia (CML) rendered drug resistant with a threonine-315-isoleucine (T315I) mutation is associated with increased incidence of vascular adverse events (VAEs). This case series sought to elucidate the mechanism of these VAEs. Methods: Two patients with CML and two patients with Philadelphia chromosome positive acute lymphocytic leukemia (Ph+ ALL) on at least one year of ponatinib therapy were imaged with optical coherence tomography (OCT) after presenting with angina. An additional four patients from an institutional OCT database were matched across clinical parameters (age, sex, diabetes, prior coronary artery disease (CAD) history, prior congestive heart failure (CHF) history, hypertension, and family history of CAD) to serve as controls. Plaque composition was assessed using operator-assisted virtual histology OCT (vOCT). Results: Clinical presentations included 2 cases of NSTEMI, 1 case of unstable angina, and 1 case of stable angina pectoris among the 4 ponatinib patients. Atherosclerosis was composed primarily of calcium and fibrous tissue with minimal lipid presence (0 ± 0°) compared to the matched controls. The matched controls had a mean non-zero lipid content of 48.43 ± 18.36°, ( P < 0.05) (Table 1). Three of the four patients had cardiovascular risk factors. Conclusions: Ponatinib causes VAEs through non-lipid plaque formation driving a symptomatology consistent with an oxygen supply-demand mismatch. This mechanism is distinct from traditional pathways of excess lipid and plaque instability causing coronary artery disease and merits further investigation.