Anthony D. Pomicter

BCR-ABL1 compound mutations in tyrosine kinase inhibitor–resistant CML: frequency and clonal relationships

BCR-ABL1 compound mutations can confer high-level resistance to imatinib and other ABL1 tyrosine kinase inhibitors (TKIs). The third-generation ABL1 TKI ponatinib is effective against BCR-ABL1 point mutants individually, but remains vulnerable to certain BCR-ABL1 compound mutants. To determine the frequency of compound mutations among chronic myeloid leukemia patients on ABL1 TKI therapy, in the present study, we examined a collection of patient samples (N = 47) with clear evidence of 2 BCR-ABL1 kinase domain mutations by direct sequencing. Using a cloning and sequencing method, we found that 70% (33/47) of double mutations detected by direct sequencing were compound mutations. Sequential, branching, and parallel routes to compound mutations were common. In addition, our approach revealed individual and compound mutations not detectable by direct sequencing. The frequency of clones harboring compound mutations with more than 2 missense mutations was low (10%), whereas the likelihood of silent mutations increased disproportionately with the total number of mutations per clone, suggesting a limited tolerance for BCR-ABL1 kinase domain missense mutations. We conclude that compound mutations are common in patients with sequencing evidence for 2 BCR-ABL1 mutations and frequently reflect a highly complex clonal network, the evolution of which may be limited by the negative impact of missense mutations on kinase function.

Combined STAT3 and BCR-ABL1 inhibition induces synthetic lethality in therapy-resistant chronic myeloid leukemia

Mutations in the BCR-ABL1 kinase domain are an established mechanism of tyrosine kinase inhibitor (TKI) resistance in Philadelphia chromosome-positive leukemia, but fail to explain many cases of clinical TKI failure. In contrast, it is largely unknown why some patients fail TKI therapy despite continued suppression of BCR-ABL1 kinase activity, a situation termed BCR-ABL1 kinase-independent TKI resistance. Here, we identified activation of signal transducer and activator of transcription 3 (STAT3) by extrinsic or intrinsic mechanisms as an essential feature of BCR-ABL1 kinase-independent TKI resistance. By combining synthetic chemistry, in vitro reporter assays, and molecular dynamics-guided rational inhibitor design and high-throughput screening, we discovered BP-5-087, a potent and selective STAT3 SH2 domain inhibitor that reduces STAT3 phosphorylation and nuclear transactivation. Computational simulations, fluorescence polarization assays and hydrogen–deuterium exchange assays establish direct engagement of STAT3 by BP-5-087 and provide a high-resolution view of the STAT3 SH2 domain/BP-5-087 interface. In primary cells from chronic myeloid leukemia (CML) patients with BCR-ABL1 kinase-independent TKI resistance, BP-5-087 (1.0 μM) restored TKI sensitivity to therapy-resistant CML progenitor cells, including leukemic stem cells. Our findings implicate STAT3 as a critical signaling node in BCR-ABL1 kinase-independent TKI resistance, and suggest that BP-5-087 has clinical utility for treating malignancies characterized by STAT3 activation.

Age-related mutations and chronic myelomonocytic leukemia

Chronic myelomonocytic leukemia (CMML) is a hematologic malignancy nearly confined to the elderly. Previous studies to determine incidence and prognostic significance of somatic mutations in CMML have relied on candidate gene sequencing, although an unbiased mutational search has not been conducted. As many of the genes commonly mutated in CMML were recently associated with age-related clonal hematopoiesis (ARCH) and aged hematopoiesis is characterized by a myelomonocytic differentiation bias, we hypothesized that CMML and aged hematopoiesis may be closely related. We initially established the somatic mutation landscape of CMML by whole exome sequencing followed by gene-targeted validation. Genes mutated in ⩾10% of patients were SRSF2, TET2, ASXL1, RUNX1, SETBP1, KRAS, EZH2, CBL and NRAS, as well as the novel CMML genes FAT4, ARIH1, DNAH2 and CSMD1. Most CMML patients (71%) had mutations in ⩾2 ARCH genes and 52% had ⩾7 mutations overall. Higher mutation burden was associated with shorter survival. Age-adjusted population incidence and reported ARCH mutation rates are consistent with a model in which clinical CMML ensues when a sufficient number of stochastically acquired age-related mutations has accumulated, suggesting that CMML represents the leukemic conversion of the myelomonocytic-lineage-biased aged hematopoietic system.

KIT signaling governs differential sensitivity of mature and primitive CML progenitors to tyrosine kinase inhibitors.

Imatinib and other BCR-ABL1 inhibitors are effective therapies for chronic myelogenous leukemia (CML), but these inhibitors target additional kinases including KIT, raising the question of whether off-target effects contribute to clinical efficacy. On the basis of its involvement in CML pathogenesis, we hypothesized that KIT may govern responses of CML cells to imatinib. To test this, we assessed the growth of primary CML progenitor cells under conditions of sole BCR-ABL1, sole KIT, and dual BCR-ABL1/KIT inhibition. Sole BCR-ABL1 inhibition suppressed mature CML progenitor cells, but these effects were largely abolished by stem cell factor (SCF) and maximal suppression required dual BCR-ABL1/KIT inhibition. In contrast, KIT inhibition did not add to the effects of BCR-ABL1 inhibition in primitive progenitors, represented by CD34(+)38(-) cells. Long-term culture-initiating cell assays on murine stroma revealed profound depletion of primitive CML cells by sole BCR-ABL1 inhibition despite the presence of SCF, suggesting that primitive CML cells are unable to use SCF as a survival factor upon BCR-ABL1 inhibition. In CD34(+)38(+) cells, SCF strongly induced pAKT(S473) in a phosphoinositide 3-kinase (PI3K)-dependent manner, which was further enhanced by inhibition of BCR-ABL1 and associated with increased colony survival. In contrast, pAKT(S473) levels remained low in CD34(+)38(-) cells cultured under the same conditions. Consistent with reduced response to SCF, KIT surface expression was significantly lower on CD34(+)38(-) compared with CD34(+)38(+) CML cells, suggesting a possible mechanism for the differential effects of SCF on mature and primitive CML progenitor cells.

shRNA library screening identifies nucleocytoplasmic transport as a mediator of BCR-ABL1 kinase-independent resistance

The mechanisms underlying tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia (CML) patients lacking explanatory BCR-ABL1 kinase domain mutations are incompletely understood. To identify mechanisms of TKI resistance that are independent of BCR-ABL1 kinase activity, we introduced a lentiviral short hairpin RNA (shRNA) library targeting ∼5000 cell signaling genes into K562(R), a CML cell line with BCR-ABL1 kinase-independent TKI resistance expressing exclusively native BCR-ABL1. A customized algorithm identified genes whose shRNA-mediated knockdown markedly impaired growth of K562(R) cells compared with TKI-sensitive controls. Among the top candidates were 2 components of the nucleocytoplasmic transport complex, RAN and XPO1 (CRM1). shRNA-mediated RAN inhibition or treatment of cells with the XPO1 inhibitor, KPT-330 (Selinexor), increased the imatinib sensitivity of CML cell lines with kinase-independent TKI resistance. Inhibition of either RAN or XPO1 impaired colony formation of CD34(+) cells from newly diagnosed and TKI-resistant CML patients in the presence of imatinib, without effects on CD34(+) cells from normal cord blood or from a patient harboring the BCR-ABL1(T315I) mutant. These data implicate RAN in BCR-ABL1 kinase-independent imatinib resistance and show that shRNA library screens are useful to identify alternative pathways critical to drug resistance in CML.

β-Catenin is required for intrinsic but not extrinsic BCR-ABL1 kinase-independent resistance to tyrosine kinase inhibitors in chronic myeloid leukemia

Activation of nuclear β-catenin and expression of its transcriptional targets promotes chronic myeloid leukemia (CML) progression, tyrosine kinase inhibitor (TKI) resistance, and leukemic stem cell self-renewal. We report that nuclear β-catenin has a role in leukemia cell-intrinsic but not -extrinsic BCR-ABL1 kinase-independent TKI resistance. Upon imatinib inhibition of BCR-ABL1 kinase activity, β-catenin expression was maintained in intrinsically resistant cells grown in suspension culture and sensitive cells cultured in direct contact (DC) with bone marrow (BM) stromal cells. Thus, TKI resistance uncouples β-catenin expression from BCR-ABL1 kinase activity. In β-catenin reporter assays, intrinsically resistant cells showed increased transcriptional activity versus parental TKI-sensitive controls, and this was associated with restored expression of β-catenin target genes. In contrast, DC with BM stromal cells promoted TKI resistance, but had little effects on Lef/Tcf reporter activity and no consistent effects on cytoplasmic β-catenin levels, arguing against a role for β-catenin in extrinsic TKI resistance. N-cadherin or H-cadherin blocking antibodies abrogated DC-based resistance despite increasing Lef/Tcf reporter activity, suggesting that factors other than β-catenin contribute to extrinsic, BM-derived TKI resistance. Our data indicate that, while nuclear β-catenin enhances survival of intrinsically TKI-resistant CML progenitors, it is not required for extrinsic resistance mediated by the BM microenvironment.

Mechanisms of resistance to the BCR-ABL1 allosteric inhibitor asciminib

Cell Lines IL-3 dependent murine Ba/F3 cells transfected with the p210 BCR-ABL1 gene as well as human chronic myeloid leukemia cell lines K562, LAMA84, KYO1 and KCL-22 were obtained from the American Type Culture Collection and grown in the recommended culture medium, RPMI 1640 media supplemented with 10% FBS, penicillin, streptomycin and 1% L-glutamine at 37°C and 5% CO2. Asciminib resistant cell lines were cultured as aforementioned with inclusion of escalating concentrations of asciminib as indicated. All human cell lines were authenticated by evaluation of short tandem repeats (STR) at 24 loci with the GenePrint 24 System (Promega). Sequencing was performed at the DNA Sequencing Core Facility, University of Utah.

Limited efficacy of BMS-911543 in a murine model of Janus kinase 2 V617F myeloproliferative neoplasm

Activation of Janus kinase 2 (JAK2), frequently as a result of the JAK2(V617F) mutation, is a characteristic feature of the classical myeloproliferative neoplasms (MPNs) polycythemia vera, essential thrombocythemia, and myelofibrosis, and it is thought to be responsible for the constitutional symptoms associated with these diseases. BMS-911543 is a JAK2-selective inhibitor that induces apoptosis in JAK2-dependent cell lines and inhibits the growth of CD34(+) progenitor cells from patients with JAK2(V617F)-positive MPN. To explore the clinical potential of this inhibitor, we tested BMS-911543 in a murine retroviral transduction-transplantation model of JAK2(V617F) MPN. Treatment was initiated at two dose levels (3 mg/kg and 10 mg/kg) when the hematocrit exceeded 70%. Following the first week, white blood cell counts were reduced to normal in the high-dose group and were maintained well below the levels in vehicle-treated mice throughout the study. However, BMS-911543 had no effect on red blood cell parameters. After 42 days of treatment, the proportion of JAK2(V617F)-positive cells in hematopoietic tissues was identical or slightly increased compared with controls. Plasma concentrations of interleukin 6, interleukin 15, and tumor necrosis factor α were elevated in MPN mice and reduced in the high-dose treatment group, whereas other cytokines were unchanged. Inhibitor activity after dosing was confirmed in a cell culture assay using the plasma of dosed mice and phosphorylated signal transducer and activator of transcription 5 flow cytometry. Collectively, these results show that BMS-911543 has limited activity in this murine model of JAK2(V617F)-driven MPN and suggest that targeting JAK2 alone may be insufficient to achieve effective disease control.

HDAC1,2 inhibition and doxorubicin impair Mre11-dependent DNA repair and DISC to override BCR-ABL1-driven DSB repair in Philadelphia chromosome-positive B-cell precursor acute lymphoblastic leukemia

Philadelphia chromosome-positive (Ph+) B-cell precursor acute lymphoblastic leukemia (ALL) expressing BCR-ABL1 oncoprotein is a major subclass of ALL with poor prognosis. BCR-ABL1-expressing leukemic cells are highly dependent on double-strand break (DSB) repair signals for their survival. Here we report that a first-in-class HDAC1,2 selective inhibitor and doxorubicin (a hyper-CVAD chemotherapy regimen component) impair DSB repair networks in Ph+ B-cell precursor ALL cells using common as well as distinct mechanisms. The HDAC1,2 inhibitor but not doxorubicin alters nucleosomal occupancy to impact chromatin structure, as revealed by MNase-Seq. Quantitative mass spectrometry of the chromatin proteome along with functional assays showed that the HDAC1,2 inhibitor and doxorubicin either alone or in combination impair the central hub of DNA repair, the Mre11–Rad51–DNA ligase 1 axis, involved in BCR-ABL1-specific DSB repair signaling in Ph+ B-cell precursor ALL cells. HDAC1,2 inhibitor and doxorubicin interfere with DISC (DNA damage-induced transcriptional silencing in cis)) or transcriptional silencing program in cis around DSB sites via chromatin remodeler-dependent and -independent mechanisms, respectively, to further impair DSB repair. HDAC1,2 inhibitor either alone or when combined with doxorubicin decreases leukemia burden in vivo in refractory Ph+ B-cell precursor ALL patient-derived xenograft mouse models. Overall, our novel mechanistic and preclinical studies together demonstrate that HDAC1,2 selective inhibition can overcome DSB repair ‘addiction’ and provide an effective therapeutic option for Ph+ B-cell precursor ALL.

Extreme mutational selectivity of axitinib limits its potential use as a targeted therapeutic for BCR-ABL1-positive leukemia

Extreme mutational selectivity of axitinib limits its potential use as a targeted therapeutic for BCR-ABL1-positive leukemia