A phase 2 trial of ponatinib in Philadelphia chromosome-positive leukemias.

BACKGROUND: Ponatinib is a potent oral tyrosine kinase inhibitor of unmutated and mutated BCR-ABL, including BCR-ABL with the tyrosine kinase inhibitor-refractory threonine-to-isoleucine mutation at position 315 (T315I). We conducted a phase 2 trial of ponatinib in patients with chronic myeloid leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-positive ALL). METHODS: We enrolled 449 heavily pretreated patients who had CML or Ph-positive ALL with resistance to or unacceptable side effects from dasatinib or nilotinib or who had the BCR-ABL T315I mutation. Ponatinib was administered at an initial dose of 45 mg once daily. The median follow-up was 15 months. RESULTS: Among 267 patients with chronic-phase CML, 56% had a major cytogenetic response (51% of patients with resistance to or unacceptable side effects from dasatinib or nilotinib and 70% of patients with the T315I mutation), 46% had a complete cytogenetic response (40% and 66% in the two subgroups, respectively), and 34% had a major molecular response (27% and 56% in the two subgroups, respectively). Responses were observed regardless of the baseline BCR-ABL kinase domain mutation status and were durable; the estimated rate of a sustained major cytogenetic response of at least 12 months was 91%. No single BCR-ABL mutation conferring resistance to ponatinib was detected. Among 83 patients with accelerated-phase CML, 55% had a major hematologic response and 39% had a major cytogenetic response. Among 62 patients with blast-phase CML, 31% had a major hematologic response and 23% had a major cytogenetic response. Among 32 patients with Ph-positive ALL, 41% had a major hematologic response and 47% had a major cytogenetic response. Common adverse events were thrombocytopenia (in 37% of patients), rash (in 34%), dry skin (in 32%), and abdominal pain (in 22%). Serious arterial thrombotic events were observed in 9% of patients; these events were considered to be treatment-related in 3%. A total of 12% of patients discontinued treatment because of an adverse event. CONCLUSIONS: Ponatinib had significant antileukemic activity across categories of disease stage and mutation status. (Funded by Ariad Pharmaceuticals and others; PACE ClinicalTrials.gov number, NCT01207440 .).

Novel pyrrolo-1,5-benzoxazepine compounds display significant activity against resistant chronic myeloid leukaemia cells in vitro, in ex vivo patient samples and in vivo.

BACKGROUND: Imatinib is a direct and potent inhibitor of the constitutively active tyrosine kinase, breakpoint cluster region-Abelson (Bcr-Abl), which is central to the pathogenesis of chronic myeloid leukaemia (CML) patients. As such, imatinib has become the front-line treatment for CML patients. However, the recent emergence of imatinib resistance, commonly associated with point mutations within the kinase domain, has led to the search for alternative drug treatments and combination therapies for CML. METHODS: In this report, we analyse the effects of representative members of the novel pro-apoptotic microtubule depolymerising pyrrolo-1,5-benzoxazepines or PBOX compounds on chemotherapy-refractory CML cells using a series of Bcr-Abl mutant cell lines, clinical ex vivo patient samples and an in vivo mouse model. RESULTS: The PBOX compounds potently reduce cell viability in cells expressing the E225K and H396P mutants as well as the highly resistant T315I mutant. The PBOX compounds also induce apoptosis in primary CML samples including those resistant to imatinib. We also show for the first time, the in vivo efficacy of the pro-apoptotic PBOX compound, PBOX-6, in a CML mouse model of the T315I Bcr-Abl mutant. CONCLUSION: Results from this study highlight the potential of these novel series of PBOX compounds as an effective therapy against CML.

European LeukemiaNet 2020 recommendations for treating chronic myeloid leukemia.

The therapeutic landscape of chronic myeloid leukemia (CML) has profoundly changed over the past 7 years. Most patients with chronic phase (CP) now have a normal life expectancy. Another goal is achieving a stable deep molecular response (DMR) and discontinuing medication for treatment-free remission (TFR). The European LeukemiaNet convened an expert panel to critically evaluate and update the evidence to achieve these goals since its previous recommendations. First-line treatment is a tyrosine kinase inhibitor (TKI; imatinib brand or generic, dasatinib, nilotinib, and bosutinib are available first-line). Generic imatinib is the cost-effective initial treatment in CP. Various contraindications and side-effects of all TKIs should be considered. Patient risk status at diagnosis should be assessed with the new EUTOS long-term survival (ELTS)-score. Monitoring of response should be done by quantitative polymerase chain reaction whenever possible. A change of treatment is recommended when intolerance cannot be ameliorated or when molecular milestones are not reached. Greater than 10% BCR-ABL1 at 3 months indicates treatment failure when confirmed. Allogeneic transplantation continues to be a therapeutic option particularly for advanced phase CML. TKI treatment should be withheld during pregnancy. Treatment discontinuation may be considered in patients with durable DMR with the goal of achieving TFR.

Novel approaches to treating advanced systemic mastocytosis.

Mastocytosis is a myeloproliferative neoplasm characterized by expansion of abnormal mast cells (MCs) in various tissues, including skin, bone marrow, gastrointestinal tract, liver, spleen, or lymph nodes. Subtypes include indolent systemic mastocytosis, smoldering systemic mastocytosis and advanced systemic mastocytosis (AdvSM), a term collectively used for the three most aggressive forms of the disease: aggressive systemic mastocytosis, mast cell leukemia, and systemic mastocytosis with an associated clonal hematological non-mast cell disease (SM-AHNMD). MC activation and proliferation is physiologically controlled in part through stem cell factor (SCF) binding to its cognate receptor, KIT. Gain-of-function KIT mutations that lead to ligand-independent kinase activation are found in most SM subtypes, and the overwhelming majority of AdvSM patients harbor the KITD816V mutation. Several approved tyrosine kinase inhibitors (TKIs), such as imatinib and nilotinib, have activity against wild-type KIT but lack activity against KITD816V. Midostaurin, a broad spectrum TKI with activity against KITD816V, has a 60% clinical response rate, and is currently the only drug specifically approved for AdvSM. While this agent improves the prognosis of AdvSM patients and provides proof of principle for targeting KITD816V as a driver mutation, most responses are partial and/or not sustained, indicating that more potent and/or specific inhibitors are required. Avapritinib, a KIT and PDGFRα inhibitor, was specifically designed to inhibit KITD816V. Early results from a Phase 1 trial suggest that avapritinib has potent antineoplastic activity in AdvSM, extending to patients who failed midostaurin. Patients exhibited a rapid reduction in both symptoms as well as reductions of bone marrow MCs, serum tryptase, and KITD816V mutant allele burden. Adverse effects include expected toxicities such as myelosuppression and periorbital edema, but also cognitive impairment in some patients. Although considerable excitement about avapritinib exists, more data are needed to assess long-term responses and adverse effects of this novel TKI.

Mutations of the BCR-ABL-kinase domain occur in a minority of patients with stable complete cytogenetic response to imatinib.

Residual leukemia is demonstrable by reverse transcriptase-polymerase chain reaction in most patients with chronic myeloid leukemia who obtain a complete cytogenetic response (CCR) to imatinib. In patients who relapse during imatinib therapy, a high rate of mutations in the kinase domain of BCR-ABL have been identified, but the mechanisms underlying disease persistence in patients with a CCR are poorly characterized. To test whether kinase domain mutations are a common mechanism of disease persistence, we studied patients in stable CCR. Mutations were demonstrated in eight of 42 (19%) patients with successful amplification and sequencing of BCR-ABL. Mutation types were those commonly associated with acquired drug resistance. Four patients with mutations had a concomitant rise of BCR-ABL transcript levels, two of whom subsequently relapsed; the remaining four did not have an increase in transcript levels and follow-up samples, when amplifiable, were wild type. BCR-ABL-kinase domain mutations in patients with a stable CCR are infrequent, and their detection does not consistently predict relapse. Alternative mechanisms must be responsible for disease persistence in the majority of patients.

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.

Zoledronate inhibits proliferation and induces apoptosis of imatinib-resistant chronic myeloid leukaemia cells.

Although imatinib mesylate has revolutionized the treatment of chronic myeloid leukaemia (CML), resistance to the drug, manifesting as relapse after an initial response or persistence of disease, remains a therapeutic challenge. In order to overcome this, alternative or additional targeting of signaling pathways downstream of Bcr-Abl may provide the best option for improving clinical response. Bisphosphonates, such as zoledronate, have been shown to inhibit the oncogenicity of Ras, an important downstream effector of Bcr-Abl. In this study, we show that zoledronate is equally effective in inhibiting the proliferation and clonogenicity of both imatinib-sensitive and -resistant CML cells, regardless of their mechanism of resistance. This is achieved by the induction of S-phase cell cycle arrest and apoptosis, through the inhibition of prenylation of Ras and Ras-related proteins by zoledronate. The combination of imatinib and zoledronate also augmented the activity of either drug alone and this occurred in imatinib-resistant CML cells as well. Since zoledronate is already available for clinical use, these results suggest that it may be an effective addition to the armamentarium of drugs for the treatment of CML.

A single nucleotide polymorphism in the coding region of ABL and its effects on sensitivity to imatinib.

We have identified a gene polymorphism (K247R) within or close to the P-loop of BCR-ABL, which leads to the substitution of arginine for lysine. We investigated the allelic frequency of K247R by screening 157 CML patients and 213 healthy blood donors with conventional sequencing, restriction enzyme digest and single strand conformational polymorphism analysis, and found the arginine allele to be rare. Three out of five CML patients with the arginine allele of K247R failed to achieve a major cytogenetic response to imatinib, suggesting that the arginine allele may have reduced sensitivity. However, despite K247R's position in or near to the P-loop, biochemical and cellular assays of imatinib and dasatinib sensitivity showed no alteration compared to wild type. Clinicians should be aware that possession of the arginine allele of K247R does not reflect a mutation that necessitates a change in the therapeutic strategy, unless there are other signs of inadequate response to drug.

Selective induction of leukemia-associated fusion genes by high-dose ionizing radiation.

There is strong clinical and epidemiological evidence that ionizing radiation can cause leukemia by inducing DNA damage. This crucial initiation event is believed to be the result of random DNA breakage and misrepair, whereas the subsequent steps, promotion and progression, must rely on mechanisms of selective pressure to provide the expanding leukemic population with its proliferative/renewal advantage. To investigate the susceptibility of human cells to external agents at the genetic recombination stage of leukemogenesis, we subjected two hematopoietic cell lines, KG1 and HL60, to high doses of gamma-irradiation. The irradiation induced the formation of fusion genes characteristic of leukemia in both cell lines, but at a much higher frequency in KG1 than in HL60. In KG1 cells, the AML1-ETO hybrid gene [associated with the t(8;21) translocation of acute myeloid leukemia] occurred significantly more often than the BCR-ABL [associated with t(9;22) chronic myeloid leukemia] or the DEK-CAN [associated with t(6;9) acute myeloid leukemia] fusion genes. These findings support the notion that ionizing radiation can directly generate leukemia-specific fusion genes but emphasize the differing susceptibility of different cell populations and the differing frequency with which the various fusion genes are formed. The selectivity observed at the primary level of gene fusion formation may explain at least in part the differential risk for development of some but not other forms of leukemia after high-dose radiation exposure.

BCR-ABL tyrosine kinase activity regulates the expression of multiple genes implicated in the pathogenesis of chronic myeloid leukemia.

The BCR-ABL chimeric protein is thought to play a central role in the pathogenesis of Philadelphia (Ph) chromosome-positive leukemias, notably chronic myeloid leukemia (CML). There is compelling evidence that malignant transformation by BCR-ABL is critically dependent on its protein tyrosine kinase (PTK) activity. As a result, multiple signaling pathways are activated in a kinase-dependent manner, and thus the activation of such pathways may affect the expression of genes that confer the malignant phenotype. In this study, we used differential display to investigate the alterations of gene expression in BV173, a CML cell line derived from lymphoid blast crisis, after exposure to ST1571, which selectively inhibits ABL PTK activity. We show that the expression of a set of 12 genes is correlated with the kinase activity and that the profile of these genes reflects mechanisms implicated in the pathogenesis of CML. Several of the genes show a consistent pattern of altered regulation in all Ph-positive lymphoid cell lines, whereas others appear to be unique to BV173 cells. We conclude that BCR-ABL PTK activity drives the expression of specific target genes that contribute to the malignant transformation of Ph-positive cells. The identification of downstream molecules with a consistent regulation pattern may provide suitable targets for therapeutic intervention in the future.

Direct relation between BCR-ABL tyrosine kinase activity and cyclin D2 expression in lymphoblasts.

Leukemia cells bearing the Philadelphia (Ph) chromosome express a Bcr-Abl fusion protein with deregulated protein tyrosine kinase (PTK) activity, which plays a central role in the malignant transformation. Many different signal transduction pathways are activated by Bcr-Abl, but little is known about their downstream targets in specific cell lineages. We show here that Ph-positive cell lines as well as primary cells derived from chronic myeloid leukemia (CML) in lymphoid blast crisis or from acute lymphoblastic leukemia (ALL) consistently express high levels of cyclin D2, whereas expression of this protein is low or absent in comparable Ph-negative lines and Ph-positive myeloid lines. Inhibition of Bcr-Abl with STI571 resulted in down-regulation of cyclin D2 and reduction of the number of cells in S phase, although complete G1 arrest was not induced. The expression of cyclin D2 in Ph-positive lymphoblasts was mediated via the phosphatidyl-inositol-3 kinase pathway. Analogous results were seen in murine BaF/3 cells transfected with a BCR-ABL expression vector. In contrast to the human cell lines, murine Baf/BCR-ABL cells exposed to STI571 inhibitor were all arrested in G1. This arrest could be abrogated by exogenous expression of cyclin D2 from a transfected cDNA construct. We conclude that a direct connection exists between Bcr-Abl PTK activity and cell cycle progression in which cyclin D2 plays a critical role. However, cell cycle progression in human Ph-positive lymphoid cells is not entirely dependent on Bcr-Abl PTK, and additional genetic lesions must be present.

A role for FOXO1 in BCR-ABL1-independent tyrosine kinase inhibitor resistance in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) patients who relapse on imatinib due to acquired ABL1 kinase domain mutations are successfully treated with second-generation ABL1-tyrosine kinase inhibitors (ABL-TKIs) such as dasatinib, nilotinib or ponatinib. However, ~40% of relapsed patients have uncharacterized BCR-ABL1 kinase-independent mechanisms of resistance. To identify these mechanisms of resistance and potential treatment options, we generated ABL-TKI-resistant K562 cells through prolonged sequential exposure to imatinib and dasatinib. Dual-resistant K562 cells lacked BCR-ABL1 kinase domain mutations, but acquired other genomic aberrations that were characterized by next-generation sequencing and copy number analyses. Proteomics showed that dual-resistant cells had elevated levels of FOXO1, phospho-ERK and BCL-2, and that dasatinib no longer inhibited substrates of the PI3K/AKT pathway. In contrast to parental cells, resistant cells were sensitive to growth inhibition and apoptosis induced by the class I PI3K inhibitor, GDC-0941 (pictilisib), which also induced FOXO1 nuclear translocation. FOXO1 was elevated in a subset of primary specimens from relapsed CML patients lacking BCR-ABL1 kinase domain mutations, and these samples were responsive to GDC-0941 treatment ex vivo. We conclude that elevated FOXO1 contributes to BCR-ABL1 kinase-independent resistance experienced by these CML patients and that PI3K inhibition coupled with BCR-ABL1 inhibition may represent a novel therapeutic approach.

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.

FISH for BCR-ABL on interphases of peripheral blood neutrophils but not of unselected white cells correlates with bone marrow cytogenetics in CML patients treated with imatinib.

Interphase fluorescence in situ hybridization (I-FISH) for the BCR-ABL translocation performed on peripheral blood (PB) white cells has been suggested as a surrogate for conventional bone marrow (BM) cytogenetics for monitoring patients with chronic myeloid leukemia (CML). I-FISH is faster, less costly, and does not require BM aspiration. For patients treated with interferon-alpha (IFN), a good correlation between the two methods has been demonstrated in several though not all studies. However, imatinib mesylate (STI571) has largely replaced IFN as the standard drug treatment for CML, raising the question if the results obtained in IFN-treated patients are applicable to patients on imatinib. We therefore compared the two methods in patients on imatinib and patients on other therapies, mainly IFN (collectively referred to as nonimatinib therapies). Our results demonstrate that the correlation between I-FISH and cytogenetics is much weaker in patients on imatinib than in patients on nonimatinib therapies. Correction of the I-FISH values for the proportion of lymphocytes barely improved the correlation, probably as a result of unpredictable proportions of Philadelphia-positive B cells. By contrast, I-FISH of PB neutrophils was much better correlated with BM cytogenetics. We conclude that I-FISH on unselected PB white cells is not suitable for monitoring patients on imatinib.

Detection of ABL kinase domain mutations with denaturing high-performance liquid chromatography.

Mutations of the ABL kinase domain (KD) are common in patients with chronic myelogenous leukemia (CML) who develop resistance to imatinib. We developed an RT-PCR-based denaturing high-performance liquid chromatography (D-HPLC) assay to detect mutations of the ABL KD. Validation experiments using mixtures of wild type and mutant amplicons showed that the D-HPLC assay could detect mutant transcripts when they represented at least 15% of the total, and was thus twice as sensitive as automated sequencing. When D-HPLC was applied to 30 cDNAs from patients with imatinib resistance that had previously been characterized for KD mutations by direct sequencing of BCR-ABL RT-PCR products, there was concordance in 97% of samples. Resequencing confirmed the original mutations in all cases. In addition, sequencing of individual clones detected a mutation in one sample that had been mutation-positive by D-HPLC but wild type by conventional sequencing. In serial samples from the same individuals, D-HPLC detected mutations as early as 260 days before hematological relapse. D-HPLC is suitable for routine clinical monitoring of CML patients for emergence of KD mutations and may be useful for optimizing therapy. Early detection of emerging mutant clones may aid in guiding decisions regarding alternative treatment options.

High Bad and Bax mRNA expression correlate with negative outcome in acute myeloid leukemia (AML).

The search for molecular markers in AML that allow prediction of outcome has recently focused on genes involved in the regulation of programmed cell death (PCD). The aim of our study was to determine whether mRNA levels of Mdm-2, Bcl-2, Bcl-x(L), Bad, and Bax are independent prognostic parameters for outcome. Transcript levels were analyzed by real-time quantitative RT-PCR in 232 samples collected either at diagnosis or following induction chemotherapy (ICT). Multivariate COX regression analysis adjusted for chemotherapy protocol, de novo vs secondary AML, and de novo vs relapsed AML indicated: (1) At diagnosis, high expression of Bad (P = 0.015) and even more so high Bax and Bad levels (P = 0.018) predicted adverse outcome, regardless of the response to ICT. In patients who subsequently failed to enter complete remission (CR), high levels of Bad, Bax and Bax high/Bad high were associated with an increased relative risk (RR) to die from tumor (RR = 5.0 for Bad, 3.4 for Bax and 6.14 for Bax high/Bad high). (2) Following ICT, high expression of Bax (P= 0.005) and high Bcl-2/Bax ratios (P = 0.004) were independent predictors of unfavorable outcome, regardless of response to ICT. We conclude that high levels of Bax and Bad correlate with poor outcome, particularly in patients who do not enter CR and may serve as prognostic markers in AML.

Dynamics of BCR-ABL mRNA expression in first-line therapy of chronic myelogenous leukemia patients with imatinib or interferon alpha/ara-C.

We sought to determine dynamics of BCR-ABL mRNA expression levels in 139 patients with chronic myelogenous leukemia (CML) in early chronic phase, randomized to receive imatinib (n=69) or interferon (IFN)/Ara-C (n=70). The response was sequentially monitored by cytogenetics from bone marrow metaphases (n=803) and qualitative and quantitative RT-PCR from peripheral blood samples (n=1117). Complete cytogenetic response (CCR) was achieved in 60 (imatinib, 87%) vs 10 patients (IFN/Ara-C, 14%) after a median observation time of 24 months. Within the first year after CCR, best median ratio BCR-ABL/ABL was 0.087%, (imatinib, n=48) vs 0.27% (IFN/Ara-C, n=9, P=0.025). BCR-ABL was undetectable in 25 cases by real-time PCR, but in only four patients by nested PCR. Median best response in patients with relapse after CCR was 0.24% (n=3) as compared to 0.029% in patients with continuous remission (n=52, P=0.029). We conclude that (i) treatment with imatinib in newly diagnosed CML patients is associated with a rapid decrease of BCR-ABL transcript levels; (ii) nested PCR may reveal residual BCR-ABL transcripts in samples that are negative by real-time PCR; (iii) BCR-ABL transcript levels parallel cytogenetic response, and (iv) imatinib is superior to IFN/Ara-C in terms of the speed and degree of molecular responses, but residual disease is rarely eliminated.

The tyrosine kinase inhibitor STI571, like interferon-alpha, preferentially reduces the capacity for amplification of granulocyte-macrophage progenitors from patients with chronic myeloid leukemia.

OBJECTIVE: To determine whether the compound STI571 (formerly known as CGP571418B), a selective inhibitor of the protein tyrosine kinase (PTK) activity of ABL and BCR-ABL proteins, preferentially reduces the capacity for amplification of granulocyte-macrophage progenitors (CFU-GM) from patients with chronic myeloid leukemia while sparing normal CFU-GM and to compare responses of CML and normal cells with STI571 and IFN-alpha. MATERIALS AND METHODS: Chronic phase CML and normal CFU-GM were grown with and without STI571, IFN-alpha, or the two agents in combination. Colonies were plucked and replated in 96-well microtiter plates. Secondary colonies were scored, and the results were expressed as the area-under-the-curve (AUC) of the distribution of secondary colony numbers per primary CFU-GM. This value gives an overall measure of the replating ability or amplification of the original CFU-GM population. RESULTS: STI571 selectively inhibits the formation of granulocyte-macrophage colony-forming cells (CFU-GM) from CML patients. It also significantly inhibits the amplification of CML CFU-GM (p = 0.002) as measured by secondary colony formation after replating primary CFU-GM colonies. In contrast, amplification of normal CFU-GM was enhanced (p = 0.001) at low concentrations (0.1 microM) of STI571 with a return to baseline at 10 microM STI571. Addition of interferon (IFN)-alpha to STI571 abolished the increase in normal CFU-GM amplification seen with either agent alone. There was a highly significant correlation between the in vitro response to STI571 and the in vitro response to IFN-alpha (r = 0.74 for CML cells, and 0.77 for normal cells). CONCLUSION: We conclude that STI571, like IFN-alpha, preferentially suppresses amplification of CML CFU-GM while sparing normal CFU-GM.

A coiled-coil mimetic intercepts BCR-ABL1 dimerization in native and kinase-mutant chronic myeloid leukemia.

Targeted therapy of chronic myeloid leukemia (CML) is currently based on small-molecule inhibitors that directly bind the tyrosine kinase domain of BCR-ABL1. This strategy has generally been successful, but is subject to drug resistance because of point mutations in the kinase domain. Kinase activity requires transactivation of BCR-ABL1 following an oligomerization event, which is mediated by the coiled-coil (CC) domain at the N terminus of the protein. Here, we describe a rationally engineered mutant version of the CC domain, called CC(mut3), which interferes with BCR-ABL1 oligomerization and promotes apoptosis in BCR-ABL1-expressing cells, regardless of kinase domain mutation status. CC(mut3) exhibits strong proapoptotic and antiproliferative activity in cell lines expressing native BCR-ABL1, single kinase domain mutant BCR-ABL1 (E255V and T315I) or compound-mutant BCR-ABL1 (E255V/T315I). Moreover, CC(mut3) inhibits colony formation by primary CML CD34(+) cells ex vivo, including a sample expressing the T315I mutant. These data suggest that targeting BCR-ABL1 with CC mutants may provide a novel alternative strategy for treating patients with resistance to current targeted therapies.

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

Activation of nuclear ß-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 ß-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, ß-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 ß-catenin expression from BCR-ABL1 kinase activity. In ß-catenin reporter assays, intrinsically resistant cells showed increased transcriptional activity versus parental TKI-sensitive controls, and this was associated with restored expression of ß-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 ß-catenin levels, arguing against a role for ß-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 ß-catenin contribute to extrinsic, BM-derived TKI resistance. Our data indicate that, while nuclear ß-catenin enhances survival of intrinsically TKI-resistant CML progenitors, it is not required for extrinsic resistance mediated by the BM microenvironment.