BCR-ABL Mutations Research Articles

PBA2, a novel inhibitor of the β-catenin/CBP pathway, eradicates chronic myeloid leukemia including BCR-ABL T315I mutation

BackgroundBCR-ABL is a constitutively active tyrosine kinase that stimulates multiple downstream signaling pathways to promote the survival and proliferation of chronic myeloid leukemia (CML) cells. The clinical application of specific BCR-ABL tyrosine kinase inhibitors (TKIs) has led to significantly improved prognosis and overall survival in CML patients compared to previous treatment regimens. However, direct targeting of BCR-ABL does not eradicate CML cells expressing T315I-mutated BCR-ABL. Our previous study revealed that inhibiting CREB binding protein (CBP) is efficacious in activating β-catenin/p300 signaling, promoting cell differentiation and inducing p53/p21-dependent senescence regardless of BCR-ABL mutation status. We hypothesize that the specific inhibition of CBP may represent a novel strategy to promote β-catenin/p300-mediated differentiation and suppress cancer cell proliferation for treating CML patients.MethodsThe anticancer efficacy of PBA2, a novel CBP inhibitor, in CML cells expressing wild-type or T315I-mutated BCR-ABL was investigated in vitro and in vivo. Cell differentiation was determined by the nitroblue tetrazolium (NBT) reduction assay. The extent of cellular senescence was assessed by senescence-associated β-galactosidase (SA-β-Gal) activity. Cytotoxicity was measured by MTS assay. RNA interference was performed to evaluate the cell proliferation effects of CBP knockdown. The interaction of β-catenin and CBP/p300 was examined by co-immunoprecipitation assay.ResultsPBA2 exhibited significantly higher anticancer effects than imatinib in CML cells harboring either wild-type or T315I-mutated BCR-ABL both in vitro and in vivo. Mechanistically, PBA2 reduced CBP expression and promoted β-catenin-p300 interaction to induce cell differentiation and senescence.ConclusionOur data supported the rational treatment of CML by inhibiting the β-catenin/CBP pathway regardless of BCR-ABL mutation status.

Celastrol induces DNA damage and cell death in BCR-ABL T315I-mutant CML by targeting YY1 and HMCES

BackgroundChronic myeloid leukemia (CML) is driven primarily by the constitutively active BCR-ABL fusion oncoprotein. Although the development of tyrosine kinase inhibitors has markedly improved the prognosis of CML patients, it remains a significant challenge to overcome drug-resistant mutations, such as the T315I mutation of BCR-ABL, and achieve treatment-free remission in the clinic. PurposeThe identification of new intervention targets beyond BCR-ABL could provide new perspectives for future research and therapeutic intervention. A network pharmacology analysis was conducted to identify the most promising natural product with anti-CML activity. Celastrol was selected for further analysis to gain insights into its mechanism of action (MoA), with the aim of identifying potential new intervention targets for BCR-ABL T315I-mutant CML. MethodsTranscriptomic and proteomic analyses were conducted to systematically investigate the molecular MoA of celastrol in K562T315I cells. To identify the target proteins of celastrol, mass spectrometry-coupled cellular thermal shift assay (MS-CETSA) was carried out, followed by validations with genetic knockdown and overexpression, cell proliferation assay, comet assay, Western blotting, celastrol probe-based in situ labeling and pull-down assay, molecular docking, and biolayer interferometry. ResultsOur multi-omics analyses revealed that celastrol primarily induces DNA damage accumulation and the unfolded protein response in K562T315I cells. Among the twelve most potential celastrol targets, experimental evidence demonstrated that the direct interaction of celastrol with YY1 and HMCES increases the levels of DNA damage, leading to cell death. ConclusionThis study represents the first investigation utilizing a proteome-wide label-free target deconvolution approach, MS-CETSA, to identify the protein targets of celastrol. This study also develops a new systems pharmacology strategy. The findings provide new insights into the multifaceted mechanisms of celastrol and, more importantly, highlight the potential of targeting proteins in DNA damage and repair pathways, particularly YY1 and HMCES, to combat drug-resistant CML.

Antibody-drug Conjugates in Cancer Treatment: An Overview

The development of antibody-drug conjugates (ADCs) has significantly impacted cancer therapy, progressing from foundational discoveries in the late 19th century to contemporary clinical applications. With the approval of the first ADC in 2000 and subsequent advancements, including over 30 ADCs in advanced clinical development, the therapeutic landscape for cancer patients has undergone a notable transformation. From initial cleavable linker technologies to the latest third-generation ADCs, continuous innovation in ADC design has been evident. Novel conjugation and linker technologies, alongside the identification of specific target antigens in solid cancers, have reinvigorated prospects for treating challenging malignancies. However, challenges such as off-target toxicity and heterogeneous antigen expression persist. The prevailing empirical approach to systemic cancer therapy administration presents challenges, including potential under-treatment of aggressive disease and over-treatment of indolent conditions, along with frequent adverse effects. Robust prognostic markers are essential to differentiate disease aggressiveness levels, guide treatment decisions, and anticipate adverse effects. Companion diagnostics for targeted therapies, such as HER-2 status for trastuzumab in breast cancer and BCR–ABL mutations for imatinib resistance in CML, enable personalized treatment strategies. Similarly, BRCA mutations predict response to PARP inhibitors in breast and ovarian cancers, while BRAF mutations guide treatment with BRAF inhibitors in melanoma. Patient selection strategies for clinical trials involving ADCs rely on prospective selection or retrospective analysis, each with its merits and challenges.

S1P Signaling Genes as Prominent Drivers of BCR-ABL1-Independent Imatinib Resistance and Six Herbal Compounds as Potential Drugs for Chronic Myeloid Leukemia.

Imatinib (IM), a breakthrough in chronic myeloid leukemia (CML) treatment, is accompanied by discontinuation challenges owing to drug intolerance. Although BCR-ABL1 mutation is a key cause of CML resistance, understanding mechanisms independent of BCR-ABL1 is also important. This study investigated the sphingosine-1-phosphate (S1P) signaling-associated genes (SphK1 and S1PRs) and their role in BCR-ABL1-independent resistant CML, an area currently lacking investigation. Through comprehensive transcriptomic analysis of IM-sensitive and IM-resistant CML groups, we identified the differentially expressed genes and found a notable upregulation of SphK1, S1PR2, and S1PR5 in IM-resistant CML. Functional annotation revealed their roles in critical cellular processes such as proliferation and GPCR activity. Their network analysis uncovered significant clusters, emphasizing the interconnectedness of the S1P signaling genes. Further, we identified interactors such as BIRC3, TRAF6, and SRC genes, with potential implications for IM resistance. Additionally, receiver operator characteristic curve analysis suggested these genes' potential as biomarkers for predicting IM resistance. Network pharmacology analysis identified six herbal compounds-ampelopsin, ellagic acid, colchicine, epigallocatechin-3-gallate, cucurbitacin B, and evodin-as potential drug candidates targeting the S1P signaling genes. In summary, this study contributes to efforts to better understand the molecular mechanisms underlying BCR-ABL1-independent CML resistance. Moreover, the S1P signaling genes are promising therapeutic targets and plausible new innovation avenues to combat IM resistance in cancer clinical care in the future.

Mutations in Genes Producing Nitric Oxide and Hydrogen Sulfide and Their Connection With Apoptotic Genes in Chronic Myeloid Leukemia.

Background Despite advances in chronic myeloid leukemia (CML) genetics, the role of nitric oxide (NO) and hydrogen sulfide (H2S) gene mutations and their relationship to apoptotic genes is unclear. Therefore, this study investigated NO- and H2S-producing genes' mutations and their interactions with apoptotic genes using Sanger sequencing and next-generation sequencing (NGS). Methodology A complete blood count (CBC) was carried out to measure the total number of white blood cells, while IL-6 levels were assessed in both control and CML patients using an ELISA technique.Sanger sequencing was used to analyzemutations in the CTH and NOS3 genes, whereas NGS was applied to examine mutations on all chromosomes. Results White blood cell (WBC) and granulocyte counts were significantly higher in CML patients compared to controls (p<0.0001), and monocyte counts were similarly higher (p<0.05). Interleukin-6 (IL-6) levels were significantly elevated in CML patients thancontrols (p<0.0001), indicating a possible link to CML etiology or progression. Multiple mutations have been identified in both genes, notably in CTH exon 12 and the NOS3genes VNTR, T786C, and G894T. This study also measured IL-6 concentrations using IL-6 assays, identifying its potential as a CML prognostic diagnostic. WBC counts, granulocyte counts, and mid-range absolute counts, or MID counts, were significantly higher in CML patients than in normal control individuals. NGS identified 1643 somatic and sex chromosomal abnormalities and 439 actively expressed genes in CML patients. The findings imply a genomic landscape beyond the BCR-ABL1 mutation in CML development compared to other databases. Conclusion In conclusion, this study advances the understanding of the genetic characteristics of CML by identifying mutations in the NO- and H2S-producing genes and their complex connections with genes involved in apoptosis. The comprehensive genetic profile obtained by Sanger sequencing and NGS provides possibilities for identifying novel targets for therapy and personalized treatments for CML, therefore contributing to developments in hematological diseases.

Abstract 6918: Ba/F3-BCR-ABL engineering cell line panel, a useful platform for novel drug discovery

Abstract In the past few years, with the great success of Imatinib, the first kinase inhibitor, Imatinib rapidly became the first-line treatment for CML (Chronic myelogenous leukemia) carrying the BCR-ABL mutation. It opened a new chapter in targeted cancer treatment. Despite the increase in overall survival allowed by imatinib, the onset of drug resistance led scientists to investigate imatinib's fine structural mechanism of action to develop new and more effective compounds against mutated forms of Bcr-Abl. 7 BCR-ABL inhibitors have been approved, they are Imatinib, Dasatinib, Nilotinib, Bosutinib, Ponatinib, Olverembatinib, and Asciminib. However, acquired resistance has been a major challenge in the treatment of CML, and some patients still experience disease progression or even death due to drug resistance. BCR-ABL kinase domain mutations are one of the important mechanisms of acquired resistance, with the T315I mutation being the most common type of drug resistance mutation, accounting for approximately 25% of drug-resistant CML cases. Patients with T315I mutations in CML are resistant to all current first- and second-generation BCR-ABL inhibitors. Asciminib is designed as an allosteric inhibitor combined with an ATP-competitive TKI agent, which may hinder the development of mutation-driven acquired resistance or restore efficacy against highly resistant BCR-ABL mutants. Our group has generated 38 Ba/F3-BCR-ABL engineering cell lines as useful models for novel drug discovery in vitro and in vivo, including wild-type BCR-ABL and inhibitors leading drug resistance mutations, such as T315I, E255, Y253, F317, F359. V299, G250, A337T, V468F, and double mutations. Each cell was validated by cell sequencing and cell efficacy verification using approved BCR-ABL inhibitors. The Ba/F3-BCR-ABL engineering cell line panel can be useful for developing and evaluating next-generation BCR-ABL inhibitors and exploring newly acquired resistance mutations in BCR-ABL. Citation Format: Guoqian Wang, Yao Peng, Yu Wang, Yao Tang, Jingxiao Xu, Jinying Ning, Feng Hao. Ba/F3-BCR-ABL engineering cell line panel, a useful platform for novel drug discovery [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 6918.

A Case of Rhegmatogenous Retinal Detachment in Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by uncontrolled proliferation of granulocytes, caused the BCR-ABL1 fusion gene. While ocular manifestations of CML are rare, the presentations can range from asymptomatic to sudden vision loss. CML associated ocular findings that have been reported include retinal hemorrhages, leukemic infiltrates, and optic disc edema, but a rhegmatogenous retinal detachment (RRD) in the setting of CML has not been described. Case report. A 21-year-old man presented with intermittent vision loss in his right eye, tinnitus in the right ear, and abdominal distension. Work up revealed significant leukocytosis, splenomegaly, and a positive BCR-ABL1 mutation. He was diagnosed with CML and started on systemic therapy. Exam of the right eye revealed a large intraocular mass. After two weeks of systemic treatment, the large elevation in the right eye had improved, allowing visualization of diffuse subretinal whitening. At follow up, an RRD secondary to an atrophic hole in an area of prior subretinal infiltrates were noted. He underwent repair with a scleral buckle. Postoperative course was complicated by redetachment with proliferative vitreoretinopathy, which led to an unrepairable detachment, despite multiple surgeries with silicone oil tamponade. Ocular findings related to CML are rare, with the lowest incidence when compared to other leukemias, and are associated with worse outcomes. Posterior segment findings include intraretinal hemorrhages, Roth spots, and retinal infiltrates. This unique case describes an RRD in CML retinopathy with an aggressive course and poor anatomical result.

A highly integrated digital PCR system with on-chip heating for accurate DNA quantitative analysis

Digital polymerase chain reaction (dPCR) is extensively used for highly sensitive disease diagnosis due to its single-molecule detection ability. However, current dPCR systems require intricate DNA sample distribution, rely on cumbersome external heaters, and exhibit sluggish thermal cycling, hampering efficiency and speed of the dPCR process. Herein, we presented the development of a microwell array based dPCR system featuring an integrated self-heating dPCR chip. By utilizing hydrodynamic and electrothermal simulations, the chip's structure is optimized, resulting in improved partitioning within microwells and uniform thermal distribution. Through strategic hydrophilic/hydrophobic modifications on the chip's surface, we effectively secured the compartmentalization of sample within the microwells by employing an overlaying oil phase, which renders homogeneity and independence of samples in the microwells. To achieve precise, stable, uniform, and rapid self-heating of the chip, the ITO heating layer and the temperature control algorithm are deliberately designed. With a capacity of 22,500 microwells that can be easily expanded, the system successfully quantified EGFR plasmid solutions, exhibiting a dynamic linear range of 105 and a detection limit of 10 copies per reaction. To further validate its performance, we employed the dPCR platform for quantitative detection of BCR-ABL1 mutation gene fragments, where its performance was compared against the QuantStudio 3D, and the self-heating dPCR system demonstrated similar analytical accuracy to the commercial dPCR system. Notably, the individual chip is produced on a semiconductor manufacturing line, benefiting from mass production capabilities, so the chips are cost-effective and conducive to widespread adoption and accessibility.

Spectrum of BCR-ABL mutations in Azerbaijanian imatinib-resistant patients with chronic myeloid leukemia.

Objective: BCR-ABL1 kinase domain (KD) mutations can lead to resistance to first- and second-generation tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML). Here, we present the first report of the spectrum of mutations in the BCR-ABL1 KD of CML patients from Azerbaijan. Materials and methods: Samples for mutation screening were obtained from patients experiencing resistance to first line TKIs or from patients in acceleration phase (AP) or blast crisis (BC) at the time of diagnosis. The cDNA region corresponding to BCR-ABL1 KD was sequenced by pyrosequencing method. The χ2 test was used to assess the association of categorical variables between mutation-positive and -negative groups. In addition, the Kaplan-Meier method was applied to generate survival curves. Results: Eight different point mutations were identified in 22 (13.4%) out of 163 CML patients experiencing resistance to TKIs. The types of mutations detected were as follows: Contact binding site mutations 50% (11), SH2 domain mutations 27.4% (six), P-loop mutations 18.1% (four), and SH3 domain mutations accounting for 4.5% (one). The most common mutation was T315I, accounting for 5% (n = 8) of all patients. Significant association was identified between BCR-ABL1 mutations and additional chromosomal aberrations as well as between the mutations and disease phases (p < 0.05). Twelve out of 22 patients with BCR-ABL1 mutations and seven out of eight with T315I were in BC. Overall survival (OS) of the patients with BCR-ABL1 mutations was significantly lower comparing to the patients with no mutation (p < 0.05) and 8 patients with T315I mutation presented OS of 0%. Conclusion: T315I was the most commonly identified BCR-ABL1 mutation in TKI-resistant CML patients of Azerbaijani origin, being associated with disease progression and poor OS.

JAK2, CALR, and MPL Mutation Profiles in Colombian patients with BCR-ABL Negative Myeloproliferative Neoplasms.

Among the chronic myeloproliferative neoplasms (MPNs) not associated with BCR-ABL mutations are polycythemia vera, primary myelofibrosis, and essential thrombocythemia. These diseases are caused by mutations in genes, such as the JAK2, MPL, and CALR genes, which participate in regulating the JAK-STAT signaling pathway. This study aimed to establish the frequencies of mutations in the JAK2, MPL, and CALR genes in a group of Colombian patients with a negative clinical diagnosis of BCR-ABL chronic myeloproliferative neoplasms. The JAK2 V617F and MPL W515K mutations and deletions or insertions in exon 9 of the CALR gene were analyzed in 52 Colombian patients with polycythemia vera, primary myelofibrosis, and essential thrombocythemia. The JAK2V617F mutation was carried by 51.9% of the patients, the CALR mutation by 23%, and the MPL mutation by 3.8%; 23% were triple-negative for the mutations analyzed. In these neoplasms, 6 mutation types in CALR were identified, one of which has not been previously reported. Additionally, one patient presented a double mutation in both the CALR and JAK2 genes. Regarding the hematological results for the mutations, significant differences were found in the hemoglobin level, hematocrit level, and platelet count among the three neoplasms. Thus, this study demonstrates the importance of the molecular characterization of the JAK2, CALR and MPL mutations in Colombian patients (the genetic context of which remains unclear in the abovementioned neoplasms) to achieve an accurate diagnosis, a good prognosis, adequate management, and patient survival.

Ponatinib-induced dilated cardiomyopathy: a case report

Ponatinib is a third-generation tyrosine kinase inhibitor with potent efficacy in the treatment of acute and chronic leukemia. Despite having an effective and sustained activity in all BCR-ABL1 mutations, it may cause cardiovascular adverse effects. Here we report a 56-year-old male patient with Chronic myeloid leukemia and no prior cardiac disorder who developed acute heart failure under ponatinib treatment. Guideline-recommended medical treatment for heart failure was initiated. Coronary angiography to exclude ischemic heart disease revealed no coronary stenosis. Cardiac magnetic resonance imaging showed findings consistent with non-ischemic dilated cardiomyopathy. After interruption of ponatinib treatment, cardiac evaluation showed improvement in left ventricular ejection fraction. Patients on Ponatinib therapy should be closely monitored for signs and symptoms of heart failure.

A Novel BCR-ABL1 Degrader, Ubx-362, Can Overcome Resistance By BCR-ABL1 Kinase Domain Mutations in Chronic Myeloid Leukemia

Background Chronic Myeloid Leukemia (CML) is a clonal hematopoietic stem cell disorder characterized by the presence of the BCR::ABL1 fusion gene. Tyrosine kinase inhibitors (TKIs) have revolutionized CML treatment, providing remarkable responses in patients. However, resistance mutations within the BCR-ABL1 kinase domain can emerge, leading to treatment failure and disease progression. Method To overcome TKI resistance, we developed a novel BCR-ABL1 degrader (UBX-362), which is a proteolysis-targeting chimera (PROTAC), and investigated its therapeutic potential using several Ba/F3 cell lines expressing various BCR-ABL1 mutations. UBX-362 is designed to target BCR-ABL1 through the ubiquitin-proteasome system-mediated degradation. Proliferation assays (IC 50) were performed using the Cell Titer-Glo assay, and western blotting was conducted to measure the degradation levels of the BCR-ABL1 protein in the tested cell lines. For the in vivo xenograft mice experiments, 5 × 10 6 K562 cells were subcutaneously injected into Balb/c nude mice. UBX-362 will be orally administered at doses of 5 or 20 mg/kg, and dasatinib or ponatinib will be administered at a dose of 20 mg/kg once daily for 21 days. Results We identified potent and orally available PROTACs, among which UBX-362 exhibited superior degradation activity against both wild-type (WT) and mutant BCR-ABL1 proteins in various Ba/F3 BCR-ABL1 kinase domain mutant-expressing cell lines. In vitro experiments showed that UBX-362 effectively degrades WT and various mutant BCR-ABL1 proteins. The levels of WT BCR-ABL1 protein were measured by western blotting, yielding a half-maximal degradation concentration of UBX-362 at ~1.78 nM. Furthermore, the DC 50 of UBX-362 against E255K, V299L, F317L, and F359C mutant BCR-ABL1 proteins were in the single-digit nM range (6.395nM, 7.679nM, 4.635nM, and 5.688nM, respectively). While UBX-362's anti-proliferative activity (~2.562nM) was not found to be superior to dasatinib (~0.378nM), it still demonstrated a strong anti-proliferative effect in the CML cell line K562 and various BCR-ABL1 mutant-expressing Ba/F3 cell lines. Specifically, the IC 50 of UBX-362 was 2.477nM in the Ba/F3 WT cell line. An in vivo anti-cancer efficacy study is currently underway in the K562 xenograft mouse model to compare its effectiveness with dasatinib or ponatinib. Conclusion The findings from this pre-clinical study show the possibility of clinical development of a BCR-ABL1 degrader. And, UBX-362 can overcome resistance in CML patients with BCR-ABL1 mutations and can be a good candidate for further clinical development.

Calr+ Myelofibrosis/BCR-ABL Chronic Myeloid Leukemia Overlap Syndrome Treated with Asciminib

Introduction Myeloproliferative neoplasms are considered mutually exclusive, and reports of co-occurring mutations are rare. Over the past 10 years, approximately two dozen case reports have been published of co-occurring BCR-ABL positive and CALR positive patients. Here we present the case of a patient who was reported to have myelofibrosis (MF)/myeloproliferative neoplasm (MPN), later developed chronic myeloid leukemia (CML), and ultimately progressed to acute myeloid leukemia (AML). This is unique in the literature reviewed, and may be the first reported case of this overlap syndrome being treated with asciminib. Case presentation A 40-year old male was diagnosed in 2018 with MF/MPN based upon a hypercellular bone marrow biopsy with diffuse reticulin fibrosis. He was negative for JAK2, but was not tested for CALR or MPL mutations. Treatment was ruxolitinib and hydroxyurea. Two years later, the patient was diagnosed with CML after being admitted for recurrent fevers, headache, and shortness of breath. At that time, a type 2 CALR mutation (CALR K385Nfs*47) was noted. He started dasatinib and decitabine, which resulted in a drop from 28.31% BCR-ABL to 0.0196% (Table 1A). However, hyperuricemia and renal failure led to a change in regimen and the patient was started on asciminib and ropeginterferon alfa-2b-njft in January 2022. By December, the patient's BCR-ABL PCR dropped below quantification, but he received a diagnosis of AML based on a bone marrow biopsy which showed 43% blasts. He received cladribine and cytarabine, but was found to have leukemic infiltration of the spinal cord five months later. The patient was referred to hospice and passed away in June 2023. Discussion To our knowledge, this is the first MPN-CML patient reported to have been treated with asciminib. This case illustrates its effectiveness in treating CML, but highlights the need for improved treatments for CALR+ MPN. Moreover, this case illustrates the importance of testing for CALR, MPL, and BCR-ABL in addition to JAK2 immediately when a patient presents with an MPN. It is unknown whether this patient's CALR mutation preceded the BCR-ABL translocation, and there is debate in the literature with respect to mutation chronology. The patient's BCR-ABL burden dropped from 28.31% to 0.0196% while the CALR burden remained around 94% over the same period (Table 1B). Thus, it is unlikely that the BCR-ABL mutation preceded the CALR mutation within the same clone. The patient's &amp;gt;90% CALR allele burden is higher than reported in the other reported cases, where the burden ranged from 10% to 68%. Furthermore, none of the other cases were reported to progress to AML, though follow up data is lacking. The heavy allelic burden in our patient may have been a contributing factor to the development of AML, as has been reported elsewhere. The patient's overall mutational profile is of interest. Type 2 CALR mutations have been reported to have less favorable prognoses than type 1 mutations. In addition to the CALR and BCR-ABL mutations, the patient had ASXL1 A861Hfs*6, EZH2 D730*, and NRAS G12V. All three mutations are associated with unfavorable outcomes in MDS, AML, and/or CML. Genomic instability has been discussed in relation to both CML and JAK2-positive MPN. Our patient's heavy mutational burden suggests CALR-positive neoplasms may also present against a background of inherent genomic instability. Conclusion We reported a case of MPN, followed by CML, and then AML. The patient was positive for CALR and BCR-ABL. He achieved a complete response in CML after targeted treatment, with asciminib consolidating the gains the patient made on earlier therapies. However, he always maintained a high CALR burden. This patient's mutational profile and disease course argue for the importance of early testing for CALR and BCR-ABL in addition to JAK2 and MPL in the setting of an MPN and for further research into the underlying causes of these overlap conditions.

Discovery of Potent and Selective Next-Generation Pan-BCR-ABL Inhibitors for the Treatment of CML and Ph+ ALL

Introduction While 6 tyrosine kinase inhibitors (TKIs) are available for patients with chronic myeloid leukemia (CML) that is newly diagnosed (imatinib, dasatinib, nilotinib, bosutinib) or refractory to prior treatments (asciminib, ponatinib), only ponatinib has pan-BCR-ABL activity, i.e., maintains potent activity against all single resistance mutations, including the difficult to inhibit T315I and E255V mutants. BCR-ABL mutations are the major known mechanism of resistance not only in CML, but also in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL), where use of ponatinib in newly diagnosed patients is yielding especially encouraging results, presumably due to its ability to suppress emergence of resistance mutations. However, toxicities observed with ponatinib often lead to dose reduction or termination that can limit the benefit of its pan-BCR-ABL profile. The suboptimal safety profile of ponatinib is consistent with its relative lack of kinome selectivity, and its potent activity against KDR, FLT3, and FGFRs (unique amongst the 6 approved BCR-ABL TKIs) could potentially contribute to its unique toxicities. We therefore set out to identify a selective, potent pan-BCR-ABL inhibitor. Methods Compound potency was determined by assessing effects on viability of Ba/F3 cell lines expressing BCR-ABL variants (wild type [WT] and 11 key mutant forms) and off-targets of interest. Preliminary assessment of broader kinome selectivity was performed by calculating the percentage of 88 kinases inhibited by &amp;gt;80% by 1 µM compound (S-score (80)). For efficacy studies, mice implanted with engineered Ba/F3 cell lines were dosed orally once daily. Results Ponatinib potently inhibited BCR-ABL WT, T315I, and E255V with IC50s of 2, 4, and 7 nM respectively, and all 9 other BCR-ABL mutants tested were inhibited with IC50s &amp;lt;4 nM. However, ponatinib also potently inhibited the activity of KDR, FLT3, and FGFR1, with IC50s of 5, 2, and 17 nM, respectively, and had a high kinome S-score (80) of 0.30. Cmpd A is a representative member of a series of compounds we have identified that have a pan-BCR-ABL inhibitory profile and substantially improved selectivity over ponatinib. Cmpd A, potently inhibited BCR-ABL WT, T315I, and E255V, with IC50s of 6, 21, and 55 nM respectively, and 8/9 other BCR-ABL mutants tested were inhibited with IC50s ≤13 nM (Y253H IC50 75 nM). But in contrast to ponatinib, Cmpd A had a high degree of selectivity over FLT3 and FGFR1 (IC50s 1898 and 3323 nM), good selectivity over KDR (IC50 420 nM), and a low kinome S-score (80) of 0.08. We next compared the efficacy and tolerability of ponatinib and Cmpd A in an efficacy model driven by E255V, a key clinical resistance mutant that both compounds inhibited less potently than T315I. At 10 mg/kg, ponatinib inhibited tumor growth by 64% compared to vehicle-treated mice and was well tolerated. At 25 mg/kg, while ponatinib induced strong tumor regression (by 82% compared to baseline), it was poorly tolerated (eg, keratinized skin in 4/6 mice). In contrast 120 mg/kg Cmpd A induced strong tumor regression, by 55%, and was well tolerated (eg, 0/6 mice had keratinized skin). Conclusions We have identified a series of next-generation pan-BCR-ABL inhibitors with substantially improved selectivity over ponatinib. In vitro and in vivo characterization of compounds with further improved selectivity profiles, and the potential to be developed for use in patients with refractory CML and newly diagnosed Ph+ ALL, will be presented.

Safety and Efficacy of Tgrx-678, a Potent BCR-ABL Allosteric Inhibitor in Patients with Tyrosine Kinase Inhibitor (TKI) Resistant/Refractory Chronic Myeloid Leukemia (CML): Preliminary Results of Phase I Study

Background: TGRX-678 is a novel allosteric inhibitor of ABL kinases, specifically targeting the ABL Myristoyl Pocket (STAMP). In vitro data supports TGRX-678 targeting the most common BCR-ABL mutations, including T315I. Here we report an open-label, first-in-human study to evaluate the safety, preliminary efficacy, and pharmacokinetic properties of orally administered TGRX-678 in patients with resistant/refractory CML. Methods: This study included dose escalation and expansion phases. During dose escalation patients with CML in chronic or accelerated phase (CP or AP) and who had failed ≥3 prior TKIs were enrolled. Patients received orally a single dose, after a 3-5 day observation, continuously daily doses ranging from 10 to 80 mg (BID) or 40 to 240 mg (QD) were given. Dose escalation followed an adaptive Bayesian logistic regression model based on dose-limiting toxicities (DLTs) occurring in cycle 1 (28 Days). Dose-expansion had three cohorts: CML-CP without T315I mutation with ≥2 prior TKIs, CML-CP with T315I with ≥1 prior TKIs, and CML-AP with ≥1 prior TKIs. The eligible patients received continuous treatments until disease progression, intolerant toxicity, consent withdrawal, or death. Results: From April 30, 2021 to July11, 2023, 95 (CP n = 58, AP n = 37) patients were treated with TGRX-678 at the BID doses including 10 mg (n = 3), 20 mg (n = 6), 40 mg (n = 5), and 80 mg (n = 3), and QD doses including 40mg (n=18), 80 mg (n = 6), 160mg (n= 4) and 240mg (n=50). 40 (42%) patients were female. The median age was 45 (range 35-52) years and the median interval from diagnosis to initial TGRX-678 treatment was 97 (range 28-136) months. Median treatment duration was 8 (range 6-13) months. At baseline, 93 (98%) patients had received ≥2 lines of prior TKI therapy, among them, 78 (82%) received ≥3 lines of prior TKIs, 44 (46%) received ≥4 lines of prior TKIs. 46 (48%) patients had ≥1 ABL mutation and 28 (30%) had T315I mutation. Of 58 patients with CML-CP, 16 (89%) achieved complete hematologic responses (CHR), 25 (43%) major cytogenetic responses (MCyR), 19 (33%) complete cytogenetic responses (CCyR), and 11 (19%) major molecular responses (MMR). Of 14 CML-CP patients with T315I mutation, 9 (100%) achieved CHR, 9 (62%) MCyR, 8 (57%) CCyR and 7 (50%) MMR. Of 35 CML-CP patients without any ABL mutations, 12 (100%) achieved CHR, 11 (31%) MCyR, 6 (17%) CCyR and 1 (3%) MMR (Fig. A). Of 9 CML-CP patients with other ABL mutations, 4 (67%) achieved CHR, 5 (56%) MCyR, 5 (56%) CCyR and 3 (33%) MMR (Fig. B). Of 37 CML-AP patients, 31 (84%) had major hematologic response, 13 (35%) MCyR, and 9 (24%) CCyR (Fig. C). There were 22 CML-CP and 23 CML-AP patients previous received 3 rd Gen TKIs (ponatinib or HQP1351) or asciminib. Of 22 CP patients, 21 (95%) achieved CHR, 4 (18%) MCyR and 3 (14%) CCyR. Of 23 CML-AP patients, 17 (74%) achieved CHR, 5 (22%) MCyR and 3 (13%) CCyR. The PK results suggested that TGRX-678 exposure (C max and AUC tau) was dose-proportional within range of 10mg~80mg BID or 40mg~240mg QD. The elimination of TGRX-678 in human plasma is slow, and the half-life (T1/2) of which is about 120 hours. At steady state, the accumulation ratios of C max and AUC tau are 3.7-8.0 and 5.4-11.8, respectively. The high accumulation ratio of TGRX-678 resulted in not only an increase of plasma exposure, but also a decrease of the gap between C max and C trough (Fig. D). 7 DLTs were observed, one case of elevated alanine aminotransferase at 20 mg BID and five cases of thrombocytopenia at 40mg QD (1), 40mg BID (2) and 80mg QD (2) and one hepatic function abnormal at 240 mg QD. However, MTD was not reached according to protocol criteria. Most treatment-related adverse events (TRAEs) were grade 1-2. AEs ≥ grade 3 that happened more than 5% were thrombocytopenia (50%), neutropenia (42%), anemia (24%) and hypertriglyceridemia (9%). In total 15 patients discontinued the study due to disease progression (5, all APs), intolerance (2), physician's decision (2), consent withdrawal (6). There was one death occurred which was not drug related. Conclusions: Clinical activity of TGRX-678 was seen in all cohorts and across TKI-resistant mutations including T315I, providing a promising treatment option for CML CP/AP patients, including those who failed ponatinib or asciminib. Its unique PK properties might bring additional benefit to patients. TGRX-678 was well tolerated in heavily pretreated CML patients. Currently two doses were selected to optimize the RP2D. Study Identifier: NCT05434312.

Derivates of 1,6-dihyadroazaazulenes as inhibitors of tyrosine kinases BCR-ABL1 wild type and mutant T315I: a molecular dynamics approach

The protein tyrosine kinase (PTK) produced by the BCR-ABL1 gene has generated significant interest in the development of inhibitors since the presence of punctual mutations causes resistance to currently approved drugs, mainly the T315I mutation has been the most difficult to address. In this work, derivatives of 1,6-dihydroazaazulenes are studied as possible inhibitors of this PTK in its wild form and the mutant T315I. The recognition of the ligands was explored through molecular docking, and the stability of the complexes and their evolution over time was studied using molecular dynamics (MD) simulations. Our results show that complexes are energetically stable and reside on the ATP binding site in all cases during the MD experiments. Interestingly, a few of our proposed ligands presented greater affinity for T315I, finding more favorable binding free energies (ΔG) than the reference drug axitinib. Furthermore, they may act as inhibitors for both isoforms. Our findings are promising because mutation of T315I does not prevent ligand recognition, as detailed in this work, which is very important to conduct further experimental research. Communicated by Ramaswamy H. Sarma

Multiomic Single-Cell Sequencing May Distinguish BCR-ABL-Mutated Acute Myeloid Leukemia (AML) from Blast Crisis Chronic Myelogenous Leukemia (BC-CML)

Introduction The existence of BCR-ABL+ AML as an entity distinct from BC-CML is controversial. The WHO recently recognized BCR-ABL+ AML, but with the caveat that it is poorly characterized (Khoury, 2022). Clinically, BCR-ABL+ AML is associated with high risk of relapse (Neuendorff, 2018). Further, acquired BCR-ABL mutations have been identified as a cause of relapse on FLT3 inhibitors (McMahon, 2019). We hypothesize that BCR-ABL+ AML can be distinguished as a distinct biologic entity from BC-CML by the observation of BCR-ABL as a sub-clonal rather than a founder mutation in leukemogenesis. Methods To test the utility of multi-omic single cell (SC) sequencing in determining tumor phylogeny and identification of BCR-ABL+ AML vs BC-CML, we analyzed bone marrow samples from 4 patients (pts): 1 relapsed AML with clinical development of a new BCR-ABL fusion at the time of relapse, and 3 newly diagnosed BC-CML (2 myeloid, 1 lymphoid). We performed high-throughput SC DNA sequencing with simultaneous cell-surface immunophenotyping and targeted RNA sequencing for BCR-ABL fusion proteins (Mission Bio Tapestri). All SC DNA sequencing included pt-specific primers for BCR-ABL DNA breakpoints, which were previously identified using nanopore-based technology for sequencing long strands of DNA. Results We analyzed 19,197 single cells (median 1995 cells/pt). Multi-omic SC technology differentiated BCR-ABL+ AML from BC-CML by confirming the fusion arose as a branch mutation from a founding leukemic clone and was not present in all leukemic populations. In Patient 1, who was sampled at time of AML relapse, we identified pathogenic FLT3 ITD and NRAS Q61K mutations. FLT3 co-mutated with either the BCR-ABL DNA breakpoint or NRAS in the same single cell and clone, while the BCR-ABL DNA breakpoint and NRAS were not co-mutated. Phylogenetic reconstruction determined FLT3 to be a founding mutation, which later branched into FLT3/BCR-ABL and FLT3/ NRAS co-mutant subclones. Further, the FLT3-ITD/BCR-ABL and FLT3-ITD/ NRAS subclones had distinct immunophenotypes. Blasts harboring the BCR-ABL DNA breakpoint were predominantly identified in the CD117+/CD33+ population (60.2% of cells) compared to CD34+ (16.4% of cells, p &amp;lt;0.001) and blasts with monocytic differentiation (19.1% of cells, p = 0.02). Notably, BCR-ABL+ cells were not identified in T cells. NRAS mutations were predominantly identified in both CD34+ and monocytic blasts. Together, these data support BCR-ABL arising late within the evolutionary history of this pt's AML. By contrast, in Patients 2 and 3 with myeloid BC-CML, the BCR-ABL DNA breakpoint and corresponding BCR-ABL fusion were detectable in all cell populations, including blasts as well as immunophenotypically normal T cells, B cells, and macrophages. In Patient 2, we identified an IDH2 R132C mutation co-mutated with BCR-ABL. Phylogenetic reconstruction determined the IDH2 mutation arose as a branch from a founding BCR-ABL+clone. Unlike the BCR-ABL breakpoint, which was present in all cell populations, the IDH2 mutation was only identified in immunophenotypically-defined CD33+/CD34+/CD117+ myeloid blasts and was absent from bystander T cells and macrophages. Finally, Patient 4had a clinical diagnosis of lymphoid BC-CML. Like Patients 2 and 3, the BCR-ABL DNA breakpoint and corresponding RNA fusion were present in all immunophenotypically-defined blast populations. Unlike Patients 2 and 3, however, BCR-ABL was absent from immunophenotypically normal CD4+ and CD8+ T cells, macrophages, and monocytes. Additional pts are needed to determine whether this pattern is consistent with all lymphoid BC-CML or whether this pt's biology may have been closer to BCR-ABL+ acute lymphoblastic leukemia (ALL). Conclusions Multi-omic SC technology can distinguish BCR-ABL+ AML from myeloid BC-CML and provide direct confirmation that lineage-restriction of BCR-ABL potentially clarifies the diagnosis of discrete Ph+ leukemias. Future studies are needed to understand the role of RT-PCR based BCR-ABL1 testing as a marker of minimal residual disease in pts with BCR-ABL+ subclones, the impact of BCR-ABL as a potential driver of AML resistance, and the utility of BCR-ABL inhibitors in leukemias with late-arising BCR-ABL+ clones. Support: U10CA180821, U10CA180882, U24CA196171; https://acknowledgments.alliancefound.org. Clinicaltrials.gov Identifiers: NCT00048958, NCT00899223, NCT00900224

Efficacy and Safety of Olverembatinib-Based Therapies in Patients with Ph/BCR-ABL1-Positive Acute Lymphoblastic Leukemia

Background Olverembatinib is an oral, third-generation BCR-ABL1 tyrosine kinase inhibitor (TKI) developed in China that could effectively target wild-type and mutant BCR-ABL1 kinase. It is effective and well-tolerated for patients with chronic myeloid leukemia. However, its efficacy and safety in patients with Ph/BCR-ABL1-Positive acute lymphoblastic leukemia are not entirely clear. This study aims to analyze the efficacy and safety of Olverembatinib-based therapies in patients with Ph/BCR-ABL1-Positive acute lymphoblastic leukemia. Methods Patients with Ph/BCR-ABL1-positive acute lymphoblastic leukemia who received the Olverembatinib-based therapies in our center were included to conduct this retrospective study. Results Totally 33 patients with Ph/BCR-ABL1-positive acute lymphoblastic leukemia were included in this study with a median age of 41 years, of whom 21 (63.63%) were female. Among them, 19 subjects who had been previously treated with standard chemotherapy and three newly diagnosed patients received Olverembatinib-based therapies, while 11 patients received Olverembatinib-based therapies after hematopoietic stem cell transplantation. After the first course of the Olverembatinib-based regimen, 33 (100%) patients experienced remission (CR/CRi/ Morphologic leukemia-free state), and 22 (66.67%) achieved the negative minimal residual disease (MRD) of BCR-ABL1/ ABL. Followed by the first course of the Olverembatinib-based regimen, five patients with negative MRD were treated with HSCT successfully and 27 received the second course of Olverembatinib-based therapy, of whom 21 (77.78%) acquired negative MRD. For six patients with negative MRD of BCR-ABL1/ ABL1, Olverembatinib-based therapies could remain the negative MRD. These patients with negative MRD will receive allogeneic hematopoietic stem cell transplantation after Olverembatinib-based therapy. Olverembatinib-based therapies were generally well tolerated. Common hematologic adverse events included leukopenia, neutropenia, anemia, and thrombocytopenia. Common nonhematologic adverse events included hepatotoxicity, nephrotoxicity, myalgia, arthralgia, rash, and edema, of which most were mild. Conclusion Olverembatinib-based therapies are generally well tolerated and efficacious for patients with Ph/BCR-ABL1-positive acute lymphoblastic leukemia.

Transcriptomic classes of BCR-ABL1 lymphoblastic leukemia

In BCR-ABL1 lymphoblastic leukemia, treatment heterogeneity to tyrosine kinase inhibitors (TKIs), especially in the absence of kinase domain mutations in BCR-ABL1, is poorly understood. Through deep molecular profiling, we uncovered three transcriptomic subtypes of BCR-ABL1 lymphoblastic leukemia, each representing a maturation arrest at a stage of B-cell progenitor differentiation. An earlier arrest was associated with lineage promiscuity, treatment refractoriness and poor patient outcomes. A later arrest was associated with lineage fidelity, durable leukemia remissions and improved patient outcomes. Each maturation arrest was marked by specific genomic events that control different transition points in B-cell development. Interestingly, these events were absent in BCR-ABL1+ preleukemic stem cells isolated from patients regardless of subtype, which supports that transcriptomic phenotypes are determined downstream of the leukemia-initialing event. Overall, our data indicate that treatment response and TKI efficacy are unexpected outcomes of the differentiation stage at which this leukemia transforms.

Concomitant BCR-ABL and JAK2 V617F in a Patient with Myeloproliferative Neoplasm: A Case Report

Myeloproliferative neoplasm (MPN) is a clonal proliferation of the haematopoietic stem cells leading to activated tyrosine kinase signaling activity. Myeloproliferative neoplasm is classified into BCR-ABL positive chronic myeloid leukemia (CML) and BCR-ABL negative MPN which harbors JAK2 V617F mutation in most cases. The genetic combination of BCR-ABL and JAK2 V617F mutation is rare with estimated frequency of 0.4% based on recent study. Herein, we reported a case of a man diagnosed with CML detected by fluorescence in-situ hybdridisation (FISH) showing atypical BCR-ABL fusion pattern in 29% nucleated cells (cut-off levels ≥5% for positive signals) in the presence of JAK2 V617F mutation. However, the BCR-ABL transcript was not detected by the conventional reverse transcriptase-polymerase chain reaction (RT-PCR) method which was specific for major fragments. Interestingly, complete hematological remission was not achieved despite initiation of tyrosine kinase inhibitor (Imatinib). In conclusion, it is imperative to scrutinise CML cases for concomitant JAK2 V617F mutation especially patients with atypical clinical or laboratory findings. Therefore, close monitoring with clinical and ancillary technique especially FISH and molecular methods such as DNA sequencing were crucial to help achieve complete hematological, cytogenetic and deep molecular response alongside targeted therapy.