BCR-ABL Kinase Research Articles

Identification of Patient-Specific Anti-Apoptotic Molecules As Therapeutic Targets in Poor Prognosis Acute Lymphoblastic Leukemia (ALL)

Relapse and refractory ALL shows dismal prognosis despite recent progress in intensive chemotherapy and development of molecular targeting agents. In this study, we aimed to identify vulnerabilities in genetically-diverse ALL and to find additional therapeutic targets in Philadelphia chromosome-positive (Ph+) ALL and chronic myeloid myeloid leukemia (CML) to overcome BCR-ABL tyrosine kinase inhibitor (TKI) resistance.To this end, we performed in vitro drug screening using 23 human ALL samples (9 Ph+ ALL, 5 MLL-ALL, 1 ALL with ETV-RUNX1, 5 B-ALL, not otherwise specified and 4 T-ALL), 10 CML samples and eight mixed phenotype acute leukemia (MPAL) samples (four B-Myeloid and four T-Myeloid). Based on our recent findings in AML (Hashimoto, Saito et al., Nature Cancer 2021), we treated leukemia-engrafting cells with small molecules targeting anti-apoptotic molecules (BCL-2, MCL-1 or BIRC) or molecules associated with cell division (AURKB) in addition to tyrosine kinase inhibitors.Among these compounds, BIRC inhibitor and venetoclax exhibited great efficacy. Responsiveness to each compound was: 28 of 41 for BIRC inhibitor (68.3%), 24 of 40 for venetoclax (60%), 11 of 37 for MCL-1 inhibitor (29.7%) and 4 of 37 for AURKB inhibitor (10.8%). We found differential sensitivity between T-ALL/MPAL T-myeloid and CML. Seven out of eight T-ALL and MPAL T-myeloid samples were highly sensitive to venetoclax (87.5%), while nine out of 10 CML samples were responsive to BIRC inhibition (90%). On the other hand, among Ph+ ALL/MPAL and Ph- B-ALL/MPAL B-myeloid samples, sensitivities to BIRC inhibitor and venetoclax were variable.To identify determinants of sensitivity to compounds, we examined the relation between mutational profile and in vitro leukemia elimination through targeted DNA sequencing for 79 lymphoid and myeloid malignancy-associated somatic mutations. Consistent with our previous study, CBL-mutated leukemia showed higher sensitivity to BIRC inhibitor (four of five cases) compared with venetoclax (two of five cases). Among genes related to RAS signaling pathway, KRAS mutations were most frequent (n=6). While five of six KRAS-mutated cases were BIRC inhibitor sensitive (83.3%), three of six cases were sensitive to venetoclax (50%). For cases with mutations in BCR-ABL1 kinase domain, the sensitivity to BIRC inhibitor and venetoclax was variable.Finally, we went on to setup in vivo experiments to elucidate if targeting the patient-specific vulnerabilities resulted in potent therapeutic efficacy against patient leukemic cells. We created patient-derived xenograft (PDX) models of 5 Ph+ ALL/MPAL, 2 Ph- B-ALL/MPAL, 2 T-ALL/MPAL and 2 CML cases. For Ph+ ALL/MPAL and CML, BIRC inhibitor and/or venetoclax combined with dexamethasone (DEX) and TKI achieved effective in vivo elimination of leukemic cells as predicted by in vitro experiments. Combination therapy showed almost complete elimination of Ph+ leukemic cells even in the presence of T315I mutation. For Ph- B-ALL/MPAL and T-ALL/MPAL, preliminary in vivo experiments showed additional inhibition of BIRC and BCL-2 resulted in more profound reduction of leukemic cells in BM compared with DEX alone (Combination: 2.1% [0.25-8] of hCD45+ leukemic cells, n=13 vs. DEX alone: 67.2% [59-76.5], n=11, median [IQR], p<0.001).We found targeting anti-apoptotic molecules in combination with DEX and/or TKI eradicated human genetically-diverse ALL, MPAL and CML cells both in vitro and in vivo. Inhibition of BIRC and BCL-2 overcame glucocorticoid resistance of Ph- ALL. Altogether, our results may offer precision medicine approach and contribute to improvement of clinical outcome in treatment-resistant ALL, CML and MPAL. DisclosuresUchida: Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Astellas Pharma Inc.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma Inc.: Honoraria. Harigae: Ono pharma: Honoraria, Other: Subsidies or Donations; Astellas Pharma: Other: Subsidies or Donations; Kyowakirin: Other: Subsidies or Donations; Janssen Pharma: Honoraria; Chugai Pharma: Honoraria; Novartis Pharma: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria.

Trial in Progress: Asciminib in Monotherapy 4 CML (AIM4CML), a Phase IIIb Study of Asciminib Monotherapy in Patients with Chronic Myeloid Leukemia in Chronic Phase without or with T315I Mutations

Trial in Progress: Asciminib in Monotherapy 4 CML (AIM4CML), a Phase IIIb Study of Asciminib Monotherapy in Patients with Chronic Myeloid Leukemia in Chronic Phase without or with T315I Mutations

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

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

MS4A3 Promotes Differentiation in Chronic Myeloid Leukemia By Enhancing Common β Chain Cytokine Receptor Endocytosis

MS4A3 Promotes Differentiation in Chronic Myeloid Leukemia By Enhancing Common β Chain Cytokine Receptor Endocytosis

A Study on BCR-ABL Kinase Domain Mutations in Chronic Myeloid Leukemia from Western India

Background: BCR-ABL kinase domain(KD) mutations accounts for 60-80% of Imatinib resistance in chronic myeloid leukemia (CML) – chronic phase (CP). Patients with CML who are receiving imatinib treatment, a mutation analysis is required to find out the resistance of imatinib as per European Leukemia Net (ELN) criteria. The present study was carried out to assess for different types of mutations responsible for resistance of imatinib treatment from Western India. Methods: In a retrospective study, the patients who were tested for imatinib resistance were analysed for IRMA testing using direct sequencing of BCR-ABL transcript by Sanger method. Results: A total of 215 patients were tested for Imatinib resistance analysis (IRMA), of which 45 (20.93%) had detectable mutations. The highest frequency of mutation recorded at T315I amino acids site, followed by M244V and G250E sites. Conclusion: The patients who were tested for IRMA showed 20.93 % positive mutations with reference to its resistance are discussed.&#x0D;

ACUTE UNDIFFERENTIATED LEUKEMIA WITH BCR-ABL TRANSLOCATION: CASE REPORT AND LITERATURE REVIEW

The 2016 WHO classification defines acute undifferentiated leukemia (AUL) as a rare type of acute leukemia (AL) without defining markers for either lymphoid or myeloid lineage commitment. Literature suggests AUL does not harbour the genetic mutations commonly seen in lymphoblastic leukemia or acute myeloid leukemia (AML) including FLT3, WT1, rearrangements of KMT2A or BCR-ABL1 and it highly expresses genes associated with poor prognosis in AML (BAALC, ERG, and MN1); therefore, AUL is often associated with dismal outcome (Arber et al. 2016). This case report depicts an unusual genetic presentation of an AUL with BCR-ABL fusion gene. A 54-year-old female patient presented to our hospital in July 2021 with fatigue and adynamia one month before. She was also found with deep vein thrombosis in the left leg and segmental pulmonary thromboembolism. Laboratory exams showed Hb 9,7 g/dL, WBC 75.77×10 9 /L (77% blasts), platelets 175×10 9 /L. Coagulogram and fibrinogen were normal. Bone marrow (BM) aspiration was hypercellular, showing 86% of agranular blasts. Flow cytometry (FC) from BM blasts exhibited expression of CD34, CD45, HLA-DR, CD33, CD38, and partial expression of CD10 and CD123. The blasts were negative for MPO, CD79a, CD19, CD7, CD3, CD13, C117, CD16, CD11b, CD64, CD15, CD14, CD300e, CD61, NG2, CD71, CD4, CD36, CD22, CD42b. Conventional cytogenetics revealed a Ph+ disease (46,XX,t(9;22)(q34q11.2)[7]/46, idem, add(4)(q35)[7]/46,XX[6]). Real-time quantitative PCR showed positive BCR-ABL with p190 transcripts. FLT3 and NPM1 were found negative. Diagnosis of AUL Ph+ was made. AML chemotherapy was started with 3+7 standard doses regimen plus imatinib 600 mg/day. On day 14th BM evaluation found 89% of blasts with the same immunophenotype as in the diagnosis. The patient presented neutropenic colitis with the need to delay rescue therapy. We searched articles in the Pubmed database using the following strategy: “Bcr-Abl Tyrosine Kinase”AND “undifferentiated leukemia”, “Philadelphia chromosome”AND “undifferentiated leukemia”between 1976 and 2021. We found two case reports that described patients with AUL and positive BCR-ABL gene. In 1983, Smadja, N et al. reported a patient with AL considered at diagnosis to be undifferentiated. Neither the diagnosis cytogenetic nor FC data was provided by the authors, and the AL classification was based only on morphological characteristics. After 17 months, the patient developed Ph+ chronic myelogenous leukemia with a complex translocation (2; 9; 22). Hattori, M et al., in 1995, described a patient that following treatment to non-Hodgkin lymphoma developed myelodysplastic syndrome (MDS) which progressed to AUL, both found with the presence of chimeric BCR-ABL transcripts. The FC showed blasts that expressed CD7 and HLA-DR, and were negative for lineage markers: CD14,CD33,CD2,CD3,CD4,CD5,CD8,CDl0,CD19,CD20 and CD41. Qasrawi, A. et al., in 2019 and 2020 performed two studies analyzing data from 24 and 1444 cases of AUL, respectively. The most common pathogenic mutations were PHF6, SRSF2, RUNX1, ASXL1, and BCOR. No BCR-ABL mutation was described. We believe that with the advent of target therapies, the prognosis of AUL can improve. The patient described has the BCR-ABL gene as a potential target to a tyrosine kinase inhibitor. Considering this is not a usual finding, we hope this report can further help treatment decisions.

New insight into the catalytic -dependent and -independent roles of METTL3 in sustaining aberrant translation in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm caused by the presence of tyrosine kinase BCR-ABL1 fusion protein, which deregulate transcription and mRNA translation. Tyrosine kinase inhibitors (TKIs) are the first-choice treatment. However, resistance to TKIs remains a challenge to cure CML patients. Here, we reveal that the m6A methyltransferase complex METTL3/METTL14 is upregulated in CML patients and that is required for proliferation of primary CML cells and CML cell lines sensitive and resistant to the TKI imatinib. We demonstrate that depletion of METTL3 strongly impairs global translation efficiency. In particular, our data show that METTL3 is crucial for the expression of genes involved in ribosome biogenesis and translation. Specifically, we found that METTL3 directly regulates the level of PES1 protein identified as an oncogene in several tumors. We propose a model in which nuclear METTL3/METTL14 methyltransferase complex modified nascent transcripts whose translation is enhanced by cytoplasmic localization of METTL3, independently from its catalytic activity. In conclusion, our results point to METTL3 as a novel relevant oncogene in CML and as a promising therapeutic target for TKI resistant CML.

Compromised anti-tumor\u2013immune features of myeloid cell components in chronic myeloid leukemia patients

Chronic myeloid leukemia (CML) is a form of myeloproliferative neoplasm caused by the oncogenic tyrosine kinase BCR-ABL. Although tyrosine kinase inhibitors have dramatically improved the prognosis of patients with CML, several problems such as resistance and recurrence still exist. Immunological control may contribute to solving these problems, and it is important to understand why CML patients fail to spontaneously develop anti-tumor immunity. Here, we show that differentiation of conventional dendritic cells (cDCs), which are vital for anti-tumor immunity, is restricted from an early stage of hematopoiesis in CML. In addition, we found that monocytes and basophils, which are increased in CML patients, express high levels of PD-L1, an immune checkpoint molecule that inhibits T cell responses. Moreover, RNA-sequencing analysis revealed that basophils express genes related to poor prognosis in CML. Our data suggest that BCR-ABL not only disrupts the “accelerator” (i.e., cDCs) but also applies the “brake” (i.e., monocytes and basophils) of anti-tumor immunity, compromising the defense against CML cells.

Flavonoid Derivatives Targeting BCR-ABL Kinase: Semisynthesis, Molecular Dynamic Simulations and Enzymatic Inhibition.

Natural products have been universally approached in the research of novel trends useful to detail the essential paths of the life sciences and as a strategy for pharmacotherapeutics. This work focuses on further modification to the 6-hydroxy-flavanone building block aiming to obtain improved BCR-ABL kinase inhibitors. Ether derivatives were obtained from Williamson synthesis and triazole from Microwave- assisted click reaction. Chemical structures were finely characterized through IR, 1H and 13C NMR and HRMS. They were tested for their inhibitory activity against BCR-ABL kinase. Two inhibitors bearing a triazole ring as a pharmacophoric bridge demonstrated the strongest kinase inhibition at IC50 value of 364 nM (compound 3j) and 275 nM (compound 3k). 6-hydroxy-flavanone skeleton can be considered as a promising core for BCR-ABL kinase inhibitors.

Inhibition of AKR1B10-mediated metabolism of daunorubicin as a novel off-target effect for the Bcr-Abl tyrosine kinase inhibitor dasatinib

Bcr-Abl tyrosine kinase inhibitors significantly improved Philadelphia chromosome-positive leukaemia therapy. Apart from Bcr-Abl kinase, imatinib, dasatinib, nilotinib, bosutinib and ponatinib are known to have additional off-target effects that might contribute to their antitumoural activities. In our study, we identified aldo–keto reductase 1B10 (AKR1B10) as a novel target for dasatinib. The enzyme AKR1B10 is upregulated in several cancers and influences the metabolism of chemotherapy drugs, including anthracyclines. AKR1B10 reduces anthracyclines to alcohol metabolites that show less antineoplastic properties and tend to accumulate in cardiac tissue. In our experiments, clinically achievable concentrations of dasatinib selectively inhibited AKR1B10 both in experiments with recombinant enzyme (Ki = 0.6 µM) and in a cellular model (IC50 = 0.5 µM). Subsequently, the ability of dasatinib to attenuate AKR1B10-mediated daunorubicin (Daun) resistance was determined in AKR1B10-overexpressing cells. We have demonstrated that dasatinib can synergize with Daun in human cancer cells and enhance its therapeutic effectiveness. Taken together, our results provide new information on how dasatinib may act beyond targeting Bcr-Abl kinase, which may help to design new chemotherapy regimens, including those with anthracyclines.

Spectrum of BCR-ABL Mutations and Treatment Outcomes in Ethiopian Imatinib-Resistant Patients With Chronic Myeloid Leukemia.

PURPOSEDespite the successes achieved in chronic myeloid leukemia (CML) with tyrosine kinase inhibitor (TKI) therapy, resistance remains an obstacle. The most common mechanism of resistance is the acquisition of a point mutation in the BCR-ABL kinase domain. Few studies have reported African patients with CML in regard to such mutations. We here report the types of BCR-ABL mutations in Ethiopian imatinib-resistant patients with CML and their outcome.PATIENTS AND METHODSPatients with CML with a diagnosis of imatinib resistance who were tested for BCR-ABL mutation between 2014 and September 2019 were included.RESULTSA total of 962 cases of CML on imatinib therapy were reviewed and 164 cases of failure were found. Of these, only 31 cases (19%) had mutation analysis performed. Most cases (94%) were secondary failures. At the time of CML diagnosis, the median age was 33 years and the majority presented with features of advanced-phase disease. Of the 31 patients, 22 mutations were found (65%). The types of mutations detected were as follows: non–P-loop mutations 36% (11), P-loop mutations 13% (four), and alternatively spliced BCR-ABL variants 23% (seven). The splice variant frequently detected was BCR-ABL35INS (20%). Twenty-six of the 31 patients (84%) were switched to second-line TKIs, whereas in four patients (13%), imatinib dose escalation was done. Overall, the outcome revealed that 16 patients (52%) were alive with complete hematologic response, whereas 12 patients (39%) had died. All patients who expressed BCR-ABL135INS were treated with second-line TKIs, and two of them (33%) had died because of disease progression.CONCLUSIONIn Ethiopia, CML affects the young and point mutations were frequently detected in imatinib-resistant patients. BCR-ABL1 35INS was also prevalent and associated with disease progression.

Effect of HSP90AB1 and CC domain interaction on Bcr-Abl protein cytoplasm localization and function in chronic myeloid leukemia cells

BackgroundThe fusion oncoprotein Bcr-Abl is mostly located in the cytoplasm, which causes chronic myeloid leukemia (CML). After moving into the nucleus, the fusion protein can induce apoptosis of CML cells. The coiled-coil domain (CC domain) of Bcr-Abl protein plays a central role in the subcellular localization. However, how CC domain affects subcellular localization of Bcr-Abl remains unclear.MethodsHerein, the key proteins interacting with the Bcr-Abl CC domain were screened by immunoprecipitation binding mass spectrometry. The specific site of Bcr-Abl CC domain binding to target protein was predicted by Deep Viewer. Immunoprecipitation assay was used to confirmed the specific sites of protein binding. IF and western blot were used to observe the subcellular localization of target protein. Western blot was used to examine the protein changes. CCK-8, clonal formation test and FCM cycle detection were used to observe the effect of inhibitor on the proliferation ability of CML cells. FCM apoptosis detection was used to observe the level of cells apoptosis.ResultsHSP90AB1 interacts with Bcr-Abl CC domain via N-terminal domain (NTD), preventing the transport of Bcr-Abl protein to the nucleus and maintaining the activation of Bcr-Abl tyrosine kinase. The nucleus-entrapped Bcr-Abl markedly inhibits the proliferation and induces apoptosis of CML cells by activating p73 and repressing the expression of cytoplasmic oncogenic signaling pathways mediated by Bcr-Abl. Moreover, the combination of 17AAG (Tanespimycin) with Leptomycin B (LMB) considerably decreased the proliferation of CML cells.ConclusionOur study provides evidence that it is feasible to transport Bcr-Abl into the nucleus as an alternative strategy for the treatment of CML, and targeting the NTD of HSP90AB1 to inhibit the interaction with Bcr-Abl is more accurate for the development and application of HSP90 inhibitor in the treatment of CML and other Bcr-Abl-addicted malignancies.F7yTRqYkfkXw_k-2eiVFmgVideo abstract

Abstract 38: Secondary fusions as a mechanism of BCR-ABL1 kinase-independent resistance in chronic myeloid leukemia

Abstract Chronic myeloid leukemia (CML) is defined by the presence of the BCR-ABL1 fusion protein, which results in constitutively active ABL1 tyrosine kinase activity. Although most chronic phase CML patients can be successfully treated with ABL1 tyrosine kinase inhibitors (TKIs), such as imatinib, up to one third of CML patients require alternative treatment. While the most common causes of TKI resistance in CML are BCR-ABL1 kinase domain mutations, many patients demonstrate BCR-ABL1 kinase-independent resistance through other poorly understood secondary molecular changes that mediate cell survival despite effective BCR-ABL1 kinase inhibition. Previous reports have described additional chromosomal rearrangements in CML patients at the time of disease transformation to blast crisis (Nucifora and Rowley, Blood 1995; Branford et al., Blood 2018), and we hypothesized that secondary fusion proteins may contribute to BCR-ABL1 kinase-independent resistance in CML. To explore this, we performed paired-end RNA sequencing on a cohort of 91 unique patients comprising three groups: BCR-ABL1 kinase-independent resistance (n=42), BCR-ABL1 kinase-dependent resistance (n=26), and newly diagnosed disease (n=23). Fusions were called using the STAR and TopHat methods, and in-frame fusion transcripts called by both methods were analyzed. We identified 11 secondary fusions which were recurrently observed among patients with BCR-ABL1 kinase-independent resistance, including both novel fusions and previously identified fusion proteins such as RUNX1-MECOM and CBFB-MYH11. Fusion breakpoint sequences were amplified via PCR in primary patient specimens at the time of resistance and confirmed by Sanger sequencing for 6 of the identified fusions: RUNX1-MECOM, CBFB-MYH11, KDM7A-MKRN1, TPM4-ACTB, TRDV2-TRAC, and ZNF292-PNRC1. To further evaluate the contribution of these fusion constructs to TKI resistance, we retrovirally co-expressed them with BCR-ABL1 in murine Ba/F3 cells and screened the cells against a panel of approved ABL1 TKIs in vitro using methanethiosulfonate (MTS)-based assays. Intriguingly, we confirmed that expression of KDM7A-MKRN1 was associated with varying degrees of decreased sensitivity to tested ABL1 TKIs, most pronouncedly for imatinib. Evaluation of drug sensitivity for additional fusions is underway and will be presented. Our findings suggest that secondary fusions, some of which are cytogenetically cryptic, beyond BCR-ABL1 are present in a subset of patients with BCR-ABL1 kinase-independent resistance and demonstrate decreased sensitivity to TKI treatment in vitro. Further characterization of the molecular mechanisms associated with these fusions in the context of BCR-ABL1 open opportunities for identifying new combination treatment strategies to overcome this type of resistance and improve outcomes for these patients. Citation Format: Evan J. Barnes, Christopher A. Eide, Daniel Bottomly, Beth Wilmot, Shannon K. McWeeney, Cristina E. Tognon, Brian J. Druker. Secondary fusions as a mechanism of BCR-ABL1 kinase-independent resistance in chronic myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 38.

Abstract NG06: Addressing scaffolding roles of oncogenic kinases with targeted protein degradation

Abstract Proteolysis Targeting Chimera (PROTAC) technology has emerged over the last two decades as a powerful tool for targeted degradation of proteins. PROTACs are heterobifunctional small molecules which co-opt the ubiquitin proteasome system to destroy oncogenic proteins. The development of PROTACs for receptor tyrosine kinases (EGFR, Her2 and c-MET), a protein family previously thought to be intractable for induced protein degradation, will be described. The use of VHL-recruiting PROTACs against this protein family reveals several advantages of degradation over inhibition alone including more potent inhibition of cell proliferation, a more durable and sustained downstream signaling response. Combined, these findings demonstrate the ability to target receptor tyrosine kinases for degradation using the PROTAC technology and outline the advantages of this degradation-based approach. Application of the PROTAC technology to targets, including RTKs, in hematological malignancies has also provided potential therapeutic approaches and enhanced understanding of scaffolding roles of oncogenic kinases. The receptor tyrosine kinase FLT-3 is frequently mutated in acute myeloid leukemia; however, traditional small molecule inhibitors suffer from limited efficacy in the clinic. A FLT-3 targeting PROTAC with sub-nanomolar antiproliferative activity and the ability to induce tumor regression in vivo will be described. Additionally, the discovery of apoptosis suppression as a kinase independent activity of FLT-3 ITD will be described. While the use of kinase inhibitors of oncoprotein BCR-Abl have enabled durable responses in patients with chronic myeloid leukemia (CML), issues of drug resistance and residual leukemic stem cells remain. An allosteric BCR-Abl PROTAC demonstrates significant advantages compared to inhibition and identified scaffolding roles for BCR-Abl which are not addressed by inhibitors but can be abrogated by protein degradation. Concurrent treated with the allosteric PROTAC and orthosteric inhibitors proved synergistic, particularly against some clinically relevant BCR-Abl kinase domain mutants. Furthermore, investigation into the kinase independent roles of BCR-Abl in patient stem cells suggest that these persistent leukemic stem cells are BCR-Abl agnostic. Citation Format: George Burslem. Addressing scaffolding roles of oncogenic kinases with targeted protein degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr NG06.

Abstract 1463: ATP-site inhibitor olverembatinib, HQP1351, enhanced the effect of allosteric inhibitor on the resistance conferred by the compound mutations of BCR-ABL

Abstract Treatment with tyrosine kinase inhibitors (TKIs) directed against the ATP-binding site of BCR-ABL promotes recovery of Ph+ leukemia. However, emergence of gatekeeper mutation T315I and compound mutants confer resistance to these TKIs. The allosteric inhibitor, asciminib, effectively inhibits BCR-ABL kinase through binding to the myristoyl-binding site. Combining asciminib with ponatinib can overcome only a subset of the resistance caused by BCR-ABL compound mutants. Olverembatinib (HQP1351) is a new generation TKI targeting BCR-ABL and currently in development for r/r CML. The purpose of this study is to evaluate whether a novel combination of olverembatinib and asciminib, targeting both ATP pocket and allosteric region of BCR-ABL protein, can promote the inhibitory effect on the kinase harboring compound mutations. A series of cell lines expressing BCR-ABL single or compound mutations were constructed based on BaF3 murine pro-B cell line. Effect of olverembatinib as a single agent or in combination with asciminib were analyzed using antiproliferation assay, Western blot and FACS assays in vitro. In vivo efficacy was evaluated using the syngeneic mouse model derived from BaF3 cells with T315I or compound mutations. The cell based antiproliferation studies demonstrated superior activity of olverembatinib toward BCR-ABL single or compound mutations with IC50 values ranging between 6-300 nM. In particular, olverembatinib was more effective than ponatinib, asciminib and other TKIs against those compound mutations. Moreover, the combination of olverembatinib with asciminib was highly effective against BCR-ABL compound mutations, especially those containing T315I. In vivo studies further revealed that co-administration of olverembatinib with asciminib resulted in significant prolongation of survival compared to single agents. Importantly, the anti-tumor effect was more potent than that of ponatinib plus asciminib in models harboring compound mutations. In terms of mechanism, the combined treatment synergistically downregulated phosphorylation of BCR-ABL and the downstream proteins CRKL and STAT5, and augmented cleavage of Caspase-3 and PARP-1, thus triggered apoptosis and subsequently enhanced the antitumor effects. Our results demonstrated that the combination of ATP binding site inhibitor olverembatinib and allosteric inhibitor have synergistic anti-tumor effect on tumor cells harboring single or compound mutations in BCR-ABL. This novel strategy may help to overcome the secondary compound mutations post the treatment with TKIs. Citation Format: Guangfeng Wang, Jing Lv, Chunyang Tang, Ping Min, Li Rui, Fei Zhang, Lvcheng Wang, Yangfeng Ge, Kejie Lian, Eric Liang, Dajun Yang, Yifan Zhai. ATP-site inhibitor olverembatinib, HQP1351, enhanced the effect of allosteric inhibitor on the resistance conferred by the compound mutations of BCR-ABL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1463.

Pharmacogenomics of Impaired Tyrosine Kinase Inhibitor Response: Lessons Learned From Chronic Myelogenous Leukemia.

The use of small molecules became one key cornerstone of targeted anti-cancer therapy. Among them, tyrosine kinase inhibitors (TKIs) are especially important, as they were the first molecules to proof the concept of targeted anti-cancer treatment. Since 2001, TKIs can be successfully used to treat chronic myelogenous leukemia (CML). CML is a hematologic neoplasm, predominantly caused by reciprocal translocation t(9;22)(q34;q11) leading to formation of the so-called BCR-ABL1 fusion gene. By binding to the BCR-ABL1 kinase and inhibition of downstream target phosphorylation, TKIs, such as imatinib or nilotinib, can be used as single agents to treat CML patients resulting in 80 % 10-year survival rates. However, treatment failure can be observed in 20-25 % of CML patients occurring either dependent or independent from the BCR-ABL1 kinase. Here, we review approved TKIs that are indicated for the treatment of CML, their side effects and limitations. We point out mechanisms of TKI resistance focusing either on BCR-ABL1-dependent mechanisms by summarizing the clinically observed BCR-ABL1-mutations and their implications on TKI binding, as well as on BCR-ABL1-independent mechanisms of resistances. For the latter, we discuss potential mechanisms, among them cytochrome P450 implications, drug efflux transporter variants and expression, microRNA deregulation, as well as the role of alternative signaling pathways. Further, we give insights on how TKI resistance could be analyzed and what could be learned from studying TKI resistance in CML in vitro.

Allosteric Bcr-Abl Inhibitor ABL001 Overcomes Bcr-Abl Tyrosine Kinase Inhibitor Resistance and Enhances MDM2 Inhibitor CGM097 Activity in Blast Crisis CML Cells

Despite Bcr-Abl tyrosine kinase inhibitors (TKIs) are highly effective in CML, development of TKI-resistance leading to blast crisis (BC) progression is still a challenge in CML therapy. This has motivated the generation of novel agents and combination strategies to overcome the resistance. ABL001 is a small molecule allosteric Bcr-Abl kinase inhibitor that occupies the myristoyl pocket, leading to a kinase autoinhibition conformation. ABL001 has entered phase I clinical trials to treat patients with chronic, accelerated, or blast phase CML that relapsed from TKIs (Hughes et al., ASH 2016). Its activity in BC CML and ability to enhance the traditional TKIs that target ATP binding site are under investigation. TP53, a key tumor suppressor gene plays a crucial role in mediating cell death in part through regulating the expression or activity of Bcl-2 family proteins. Although highly mutated in solid tumors, the mutation rate of TP53 is relatively low in CML. However, p53 antagonist, MDM2 can be regulated by Bcr-Abl signaling and ARF, which inhibits MDM2 is frequently deleted in BC CML suggesting therapeutic potentials of inhibition of MDM2 in BC CML. Indeed, activation of p53 by MDM2 inhibition has been shown to enhance the toxicity of TKIs in BC CML (Carter et al., Oncotarget 2015, 6(31):30487). In this study, we examined the activity of ABL001 in CML cells and investigated if ABL001 enhances the activity of TKIs and synergizes with MDM2 inhibitor CGM097 in samples from heavily treated TKI resistant BC CML patients.BaF3 cells expressing BCR-ABL with or without mutations, human CML cell lines (KBM5 and KBM5-STI-T315I), and peripheral blood mononuclear cells (PBMC) isolated from primary BC CML patient samples were treated with various doses of ABL001, CGM097 or Nilotinib alone or in combination. Apoptosis was determined by Annexin V/7-AAD staining in cell lines and CD45+ (bulk), CD34+CD38+ and CD34+CD38- cells of CML patient PBMC samples. EC50 was calculated using CompuSyn or Graphpad. Protein levels were examined by Western blotting.We found that ABL001 induced apoptosis in cell lines expressing BCR-ABL (BaF3p210 and KBM5) or mutant BCR-ABL (BaF3p210E255K and KBM5-STI-T315I) but had limited activity in BaF3p210T315I cells. Importantly, ABL001 induced apoptosis in bulk, CD34+CD38+, or/and CD34+CD38- cell populations at low nM concentrations in 5 out of 6 BC CML patient samples tested. Patient samples that responded to ABL001 had either T315I and/or E255 mutations in BCR-ABL kinase domain. ABL001 inhibited Bcr-Abl signaling as measured by the levels of p-Bcr-Abl/p-Stat5/p-Crkl in BCR-ABL positive cells with or without BCR-ABL mutations. ABL001 showed higher activity than Nilotinib in 4 out of 5 responding patient samples. Nilotinib did not significantly enhance ABL001-induced apoptosis in the tested BC CML patient samples and CML cell lines. Inhibition of MDM2 with CGM097 activated p53 and induced apoptosis in BC CML patient samples. The activity of ABL001 was enhanced by CGM097 in the majority of patient samples which did not have p53 mutations. ABL001 combined with CGM097 was more effective than the combination of nilotinib with CGM097. The combinations of TKI and CGM097 were more effective in killing leukemia cells than each agent alone even when leukemia cells were co-cultured with MSCs. Neither ABL001 or CGM097 alone, nor the combination showed toxicity on normal bone marrow stem/progenitor cells.In conclusion, our results demonstrate that the allosteric Bcr-Abl Inhibitor ABL001 overcomes TKI-resistance and enhances MDM2 inhibitor CGM097 activity in BC CML cells. The combination of ABL001 and CGM097 warrants clinical evaluation. DisclosuresCarter:novartis: Research Funding; Daiichi Sankyo: Research Funding.

Expression Levels of PD-1 on CD8+ T Cells in Chronic Myeloid Leukemia with and without BCR-ABL1 kinase Mutation

Background: There are several ways for malignant cells to increase their number. One of them is to use immune checkpoints protecting malignant cells from immune-mediated injury. Upregulation of immune checkpoints contributes immune escape and tumor growth. Tyrosine kinase inhibitors (TKI) have improved the outcome of chronic myeloid leukemia (CML). TKI is generally known to inhibit the target BCR-ABL1 kinase in leukemic cells. However, constantly growing evidence suggests that some TKIs activate the immune system to exert the direct cytotoxic effects. In this study, we evaluated expression levels of PD-1 on T-cells in CML patients, and found the relation with ABL kinase mutation. We analyzed the relation between the quantitative levels of BCR-ABL and PD-1 expression percentages.Methods: We obtained 82 bone marrow (BM) samples from patients diagnosed as CML at Asan medical center in Korea between May 2016 and May 2017. The samples included 22 at diagnosis (20 in chronic phase; CP and 2 in blast phase; BP) and 60 at follow-up (52 in complete hematologic remission; CHR, 1 in CP, 1 in accelerated phase; AP, and 6 in BP). All patients at follow-up were treated with Imatinib, Dasatinib, Nilotinib and/or Radotinib. Normal BM samples were collected from 23 individuals with no evidence of hematologic malignancies as the controls. Expression of PD-1 on T cells was measured using flow cytometric analysis.Results: The median expression level of PD-1 on CD8+ T cells for the patients in CHR was 21.25%, which was significantly lower than those in controls, in CP and in BP (Fig. 1). At diagnosis of CML, the median expression level of PD-1 on CD8+ T cells was 34.7%. In CML patients treated with Imatinib and/or dasatinib, the expression levels of PD-1 on CD8+ T cells were lower than at diagnosis (respectively, P < 0.001 and P= 0.045). In CML patients treated with Nilotinib, there was no difference of expression levels of PD-1 compared with those at diagnosis. In CML patients with ABL kinase mutation, the expression levels of PD-1 on CD8+ T cells was much higher than those of CML patients without ABL kinase mutation group (P= 0.008) (Fig. 2). The level of PD-1 expression positively correlated with the quantitative levels of BCR/ABL detected by RT-PCR in BM (P= 0.024, rho = 0.270). We found 2 patients who had more than 4 follow-up data over a 6 month-period and serialized the changes of BCR/ABL quantitation, PD-1 expression percentages on CD8+ T cells and disease phases of them. PD-1 expression percentages seemed to reflect the progression of the diseases, based on very limited data (Fig. 3).Conclusion: The low expression PD-1 on CD8+ T cells might play a role in maintaining CHR in CML. The high expression of PD-1 expression in CML patients with ABL kinase mutation demonstrated that TKI had the effect of lowering the expression of PD-1 on CD8+ T cells. We conclude that PD-1 on CD8+ T cells could be important to control CML, and the immune checkpoint inhibitors might be effective in CML patients, particularly those with ABL kinase mutation. [Display omitted] DisclosuresLee:Boehringer Ingelheim: Research Funding.

Ca2+ Oscillations and NFATC1 -Activation Enable Oncogenic Signaling in Pre-B Cell Transformation and Leukemogenesis

Background: Nuclear factor of activated T cells (NFATC1) regulates activation and differentiation of B and T cells. Activation of NFATC1 requires sustained Ca2+ influx by store-operated Ca2+ entry (SOCE). Our group previously showed that oncogenic signaling from the BCR-ABL1 kinase in Ph+ ALL results in autonomous oscillatory Ca2+ signaling (Klein et al., J Exp Med 2004; Feldhahn et al., J Exp Med 2005). However, the significance of Ca2+ oscillations remained unclear. Here we examined the function of Orai and Stim1/2, the central mediators of SOCE, and the role of Ca2+ oscillations in the survival of BCR-ABL1 -driven pre-B ALL cells.Results: To investigate the consequence of inducible deletion of SOCE-effector genes, Orai1, Stim1, and Stim2, we have generated BCR-ABL1 - and CreERT2-transduced murine pre-B Orai1f/f and Stim1/2f/f cells. Both Orai1 - and Stim1/2 -deletion impaired Ca2+ influx from the extracellular space when Ca2+ uptake from cytosol to endoplasmic reticulum was pharmacologically inhibited. Consistently, both Orai1 - and Stim1/2 -deletion significantly attenuated the amplitude and frequency of autonomous Ca2+ oscillations, compared with that of empty vector controls. To determine whether the impaired Ca2+ signaling associated with cell survival and proliferation, we next analyzed their growth and colony-forming capacity. As expected, both Orai1 - and Stim1/2 -deleted pre-B ALL cells exhibited growth disadvantage and reduced colony-forming capacity, compared with empty vector controls. Flow cytometry studies of Annexin V revealed that both Orai1 - and Stim1/2 -deletion induced apoptosis in the BCR-ABL1 -transformed cells. These results suggest that autonomous Ca2+ oscillations are needed to circumvent apoptosis, and enhance cell growth and self-renewal in BCR-ABL-driven ALL cells. Interestingly, even in normal pre-B cells and in the absence of autonomous Ca2+ oscillations, both Orai1 - and Stim1/2 -deletion caused growth disadvantage suggesting the existence of similar cellular function to Ca2+ oscillations in normal pre-B cells. Subsequently, we studied NFATc1 activity by Western blot, as reflected by nuclear localization, in pre-B ALL cells before and after Orai1 - and Stim1/2 -deletion. NFATc1 is known as one of a central NFAT family member that is activated in lymphocytes upon BCR and TCR stimulation. While Orai1fl/fl and Stim1/2fl/fl B-lineage ALL cells transduced with empty vector controls displayed marked nuclear accumulation of NFATc1, Cre-mediated deletion of Orai1 and Stim1/2 strongly reduced nuclear accumulation of NFATc1, with slight increases in the cytoplasmic fraction. To examine the consequences of NFATc1 inhibition, we generated BCR-ABL1 - and CreERT2-transduced murine pre-B Nfatc1f/f and observed its effect on cell growth. As expected, inducible ablation of Nfatc1 resulted in rapid depletion of pre-B ALL cells from the cell culture compared to empty vector controls. Importantly, a NFAT-calcineurin association inhibitor, INCA-6, displayed growth inhibitory effects in all six patient-derived xenograft of B cell ALL tested, including samples from patients who relapsed after initially successful chemotherapy. These results suggest that the Ca2+-NFAT signal may suppress apoptosis, and promote drug-resistance and relapse in human pre-ALL cells.Conclusions: Taken together, our findings showed that SOCE has a critical role in NFAT activation and survival of BCR-ABL1 -driven pre-B ALL cells. Orai1- and Stim1/2-mediated autonomous Ca2+ oscillatory signal is required for BCR-ABL1-dependent proliferation. This signal may bypass the NFAT activation which suppress apoptosis and enhance proliferation in BCR-ABL-driven ALL cells. DisclosuresNo relevant conflicts of interest to declare.

Loss of G0S2 in Kinase-Independent TKI Resistance and Blastic Transformation of CML

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 have turned chronic myeloid leukemia (CML) from a fatal to a chronic condition. Despite impressive results, resistance is a clinical problem, and TKIs do not target the CML leukemic stem cell (LSC) population, which is independent of BCR-ABL1 kinase activity. To identify mechanisms associated with BCR-ABL1 kinase-independent TKI resistance, we compared the transcriptional profile of TKI-naïve CD34+ cells from newly diagnosed CML patients who subsequently either responded (n=41) or did not respond (n=18) to imatinib over a twelve-month period (McWeeney et al. Blood 2010). The transcriptional profile of imatinib resistance in non-responders revealed striking overlap with reported signatures of CML blastic transformation and also implicated pathways involved in LSC survival. These findings suggest that similar biological mechanisms may be driving three different scenarios of BCR-ABL1-independent resistance: 1) primary kinase-independent TKI resistance, 2) transformation of CML from the chronic phase (CP-CML) to the blastic phase (BP-CML), and 3) persistence of residual leukemia in TKI-responsive patients.Among the genes most profoundly downregulated in patients destined to fail imatinib therapy was G0S2 (>3-fold, p<0.02), encoding a small protein that regulates multiple cellular functions, including apoptosis, quiescence, lipid metabolism, and oxidative phosphorylation. We compared G0S2 mRNA expression in CD34+ cells from normal cord blood (n=5) and from patient specimens lacking BCR-ABL1 kinase domain mutations, including newly diagnosed CP-CML (n=6), TKI-resistant CP-CML (n=2), and BP-CML (n=5). G0S2 was 4-fold downregulated in newly diagnosed CP-CML compared to normal cord blood CD34+ cells (p<0.01), and further downregulated by >3-fold in TKI-resistant samples and BP-CML (p<0.01), consistent with published microarray data (McWeeney et al. Blood 2010). Retrospective analysis of survival data for patients whose samples were evaluated in the transcriptional profile revealed that low G0S2 expression correlated with a shorter overall survival (n=35, p=0.036). Consistently, G0S2 mRNA and protein were downregulated in TKI-resistant K562R cells lacking BCR-ABL1 kinase domain mutations compared to TKI-sensitive controls. Ectopic expression of Flag-tagged G0S2 (G0S2-Flag) significantly reduced colony formation and increased apoptosis of TKI-sensitive and TKI-resistant K562 cells, and also CD34+ cells from CP-CML (n=5, p<0.05) and BP-CML (n=3, p<0.05) patients, having a more pronounced effect in BP-CML in the presence of imatinib. G0S2-Flag did not affect colony formation or apoptosis of normal cord blood CD34+ cells (n=2), which already express high levels of G0S2. Importantly, qRT-PCR revealed that G0S2 mRNA was significantly lower in primitive CD34+38- stem cells compared to more committed CD34+38+ progenitor cells in CP-CML (n=6, p<0.01) but not normal cord blood CD34+ cells (n=4, p=ns). These data suggest that loss of G0S2 plays a role not only in primary TKI resistance and blastic transformation of CML, but also in TKI resistance of the CML LSC. Consistently, CFSE staining revealed that G0S2 mRNA levels in CP-CML CD34+ cells are lowest in cell populations with the least number of cell divisions.Altogether, these findings suggest that G0S2 plays a role in regulating leukemic stem and progenitor cell survival and TKI response in vitro, and that restoring G0S2 expression combined with BCR-ABL1 inhibition may be a novel strategy to induce treatment-free remission in CML patients with kinase-independent TKI resistance and BP-CML, or to eradicate the CML LSC population. Future studies will assess the functional role of G0S2 in LSC survival and BCR-ABL1 leukemogenesis in vivo, and determine the mechanism by which G0S2 is downregulated in TKI resistance. DisclosuresDeininger:BMS: Consultancy, Research Funding; Ariad Pharmaceuticals, Bristol Myers Squibb, CTI BioPharma Corp, Gilead, Incyte, Novartis, Pfizer, Celgene, Blue Print, Galena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy; Celgene: Research Funding; Gilead: Research Funding; ARIAD: Consultancy.