Overcoming clinical BCR-ABL1 compound mutant resistance with combined ponatinib and asciminib therapy.

BCR-ABL1 compound mutations can lead to resistance to ABL1 inhibitors in chronic myeloid leukemia (CML), which could be targeted by combining the ATP-site inhibitor ponatinib and the allosteric inhibitor asciminib. Here, we report the clinical validation of this approach in a CML patient, providing a basis for combination therapy to overcome such resistance.

TNK1 is a ubiquitin-binding and 14-3-3-regulated kinase that can be targeted to block tumor growth.

TNK1 is a non-receptor tyrosine kinase with poorly understood biological function and regulation. Here, we identify TNK1 dependencies in primary human cancers. We also discover a MARK-mediated phosphorylation on TNK1 at S502 that promotes an interaction between TNK1 and 14-3-3, which sequesters TNK1 and inhibits its kinase activity. Conversely, the release of TNK1 from 14-3-3 allows TNK1 to cluster in ubiquitin-rich puncta and become active. Active TNK1 induces growth factor-independent proliferation of lymphoid cells in cell culture and mouse models. One unusual feature of TNK1 is a ubiquitin-association domain (UBA) on its C-terminus. Here, we characterize the TNK1 UBA, which has high affinity for poly-ubiquitin. Point mutations that disrupt ubiquitin binding inhibit TNK1 activity. These data suggest a mechanism in which TNK1 toggles between 14-3-3-bound (inactive) and ubiquitin-bound (active) states. Finally, we identify a TNK1 inhibitor, TP-5801, which shows nanomolar potency against TNK1-transformed cells and suppresses tumor growth in vivo.

SIRT5 IS A DRUGGABLE METABOLIC VULNERABILITY IN ACUTE MYELOID LEUKEMIA.

We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype-agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML.

Femoral Heads from Total Hip Arthroplasty as a Source of Adult Hematopoietic Cells.

Normal human bone marrow cells are critical for studies of hematopoiesis and as controls to assess toxicity. As cells from commercial vendors are expensive, many laboratories resort to cancer-free bone marrow specimens obtained during staging or to umbilical cord blood cells, which may be abnormal or reflect a much younger age group compared to the disease samples under study. We piloted the use of femoral heads as an alternative and inexpensive source of normal bone marrow. Femoral heads were obtained from 21 successive patients undergoing elective hip arthroplasty. Mononuclear cells (MNCs) were purified with Ficoll, and CD3+, CD14+, and CD34+ cells were purified with antibody-coated microbeads. The median yield of MNCs was 8.95 × 107 (range, 1.62 × 105-2.52 × 108), and the median yield of CD34+ cells was 1.40 × 106 (range, 3.60 × 105-9.90 × 106). Results of downstream applications including qRT-PCR, colony-forming assays, and ex vivo proliferation analysis were of high quality and comparable to those obtained with standard bone marrow aspirates. We conclude that femoral heads currently discarded as medical waste are a cost-efficient source of bone marrow cells for research use.

Dasatinib overcomes stroma-based resistance to the FLT3 inhibitor quizartinib using multiple mechanisms.

FLT3-ITD mutations occur in 20-30% of AML patients and are associated with aggressive disease. Patients with relapsed FLT3-mutated disease respond well to 2nd generation FLT3 TKIs but inevitably relapse within a short timeframe. In this setting, until overt relapse occurs, the bone marrow microenvironment facilitates leukemia cell survival despite continued on-target inhibition. We demonstrate that human bone marrow derived conditioned medium (CM) protects FLT3-ITD+ AML cells from the 2nd generation FLT3 TKI quizartinib and activates STAT3 and STAT5 in leukemia cells. Extrinsic activation of STAT5 by CM is the primary mediator of leukemia cell resistance to FLT3 inhibition. Combination treatment with quizartinib and dasatinib abolishes STAT5 activation and significantly reduces the IC50 of quizartinib in FLT3-ITD+ AML cells cultured in CM. We demonstrate that CM protects FLT3-ITD+ AML cells from the inhibitory effects of quizartinib on glycolysis and that this is partially reversed by treating cells with the combination of quizartinib and dasatinib. Using a doxycycline-inducible STAT5 knockdown in the FLT3-ITD+ MOLM-13 cell line, we show that dasatinib-mediated suppression of leukemia cell glycolytic activity is STAT5-independent and provide a preclinical rationale for combination treatment with quizartinib and dasatinib in FLT3-ITD+ AML.

JAK2 ex13InDel drives oncogenic transformation and is associated with chronic eosinophilic leukemia and polycythemia vera.

The V617F mutation in the JH2 domain of Janus kinase 2 (JAK2) is an oncogenic driver in several myeloproliferative neoplasms (MPNs), including essential thrombocythemia, myelofibrosis, and polycythemia vera (PV). Other mutations in JAK2 have been identified in MPNs, most notably exon 12 mutations in PV. Here, we describe a novel recurrent mutation characterized by a common 4-amino-acid deletion and variable 1-amino-acid insertion (Leu583-Ala586DelInsSer/Gln/Pro) within the JH2 domain of JAK2. All 4 affected patients had eosinophilia, and both patients with Leu583-Ala586DelInsSer fulfilled diagnostic criteria of both PV and chronic eosinophilic leukemia (CEL). Computational and functional studies revealed that Leu583-Ala586DelInsSer (herein referred to as JAK2ex13InDel) deregulates JAK2 through a mechanism similar to JAK2V617F, activates signal transducer and activator of transcription 5 and extracellular signal-regulated kinase, and transforms parental Ba/F3 cells to growth factor independence. In contrast to JAK2V617F, JAK2ex13InDel does not require an exogenous homodimeric type 1 cytokine receptor to transform Ba/F3 cells and is capable of activating ß common chain family cytokine receptor (interleukin-3 receptor [IL-3R], IL-5R, and granulocyte-macrophage colony stimulating factor receptor) signaling in the absence of ligand, with the maximum effect observed for IL-5R, consistent with the clinical phenotype of eosinophilia. Recognizing this new PV/CEL-overlap MPN has significant clinical implications, as both PV and CEL patients are at high risk for thrombosis, and concomitant cytoreduction of red cells, neutrophils, and eosinophils may be required for prevention of thromboembolic events. Targeted next-generation sequencing for genes recurrently mutated in myeloid malignancies in patients with unexplained eosinophilia may reveal additional cases of Leu583-Ala586DelInsSer/Gln/Pro, allowing for complete characterization of this unique MPN.

The transcriptome of CMML monocytes is highly inflammatory and reflects leukemia-specific and age-related alterations.

Chronic myelomonocytic leukemia (CMML) is an aggressive myeloid neoplasm of older individuals characterized by persistent monocytosis. Somatic mutations in CMML are heterogeneous and only partially explain the variability in clinical outcomes. Recent data suggest that cardiovascular morbidity is increased in CMML and contributes to reduced survival. Clonal hematopoiesis of indeterminate potential (CHIP), the presence of mutated blood cells in hematologically normal individuals, is a precursor of age-related myeloid neoplasms and associated with increased cardiovascular risk. To isolate CMML-specific alterations from those related to aging, we performed RNA sequencing and DNA methylation profiling on purified monocytes from CMML patients and from age-matched (old) and young healthy controls. We found that the transcriptional signature of CMML monocytes is highly proinflammatory, with upregulation of multiple inflammatory pathways, including tumor necrosis factor and interleukin (IL)-6 and -17 signaling, whereas age per se does not significantly contribute to this pattern. We observed no consistent correlations between aberrant gene expression and CpG island methylation, suggesting that proinflammatory signaling in CMML monocytes is governed by multiple and complex regulatory mechanisms. We propose that proinflammatory monocytes contribute to cardiovascular morbidity in CMML patients and promote progression by selection of mutated cell clones. Our data raise questions of whether asymptomatic patients with CMML benefit from monocyte-depleting or anti-inflammatory therapies.

BCR-ABL1 tyrosine kinase inhibitor K0706 exhibits preclinical activity in Philadelphia chromosome-positive leukemia.

BCR-ABL1 tyrosine kinase inhibitors (TKIs) are the cornerstone of treatment in chronic myeloid leukemia. Although there are now four TKIs approved for use in the front-line setting, acquired TKI resistance via secondary kinase domain mutations remains a problem for patients. K0706 is a novel BCR-ABL1 TKI currently under clinical investigation with structural elements similar to those of ponatinib and dasatinib. In this article, we functionally characterize the anti-leukemic activity of K0706 using cell proliferation assays in conjunction with drug resistance screening. We provide details from molecular modeling to support our in vitro findings and additionally describe our limited clinical experience with this drug in two patients treated on trial. We demonstrate that although K0706 retains efficacy against a large spectrum of clinically relevant mutations, it does not appear to have activity against BCR-ABL1T315I. Early trial experience suggests excellent tolerability, which may positively affect the place of K0706 within the ever-expanding chronic myeloid leukemia treatment paradigm.

Combining the Allosteric Inhibitor Asciminib with Ponatinib Suppresses Emergence of and Restores Efficacy against Highly Resistant BCR-ABL1 Mutants.

BCR-ABL1 point mutation-mediated resistance to tyrosine kinase inhibitor (TKI) therapy in Philadelphia chromosome-positive (Ph+) leukemia is effectively managed with several approved drugs, including ponatinib for BCR-ABL1T315I-mutant disease. However, therapy options are limited for patients with leukemic clones bearing multiple BCR-ABL1 mutations. Asciminib, an allosteric inhibitor targeting the myristoyl-binding pocket of BCR-ABL1, is active against most single mutants but ineffective against all tested compound mutants. We demonstrate that combining asciminib with ATP site TKIs enhances target inhibition and suppression of resistant outgrowth in Ph+ clinical isolates and cell lines. Inclusion of asciminib restores ponatinib's effectiveness against currently untreatable compound mutants at clinically achievable concentrations. Our findings support combining asciminib with ponatinib as a treatment strategy for this molecularly defined group of patients.

ddeeper Than Deep: Can ddPCR Predict Successful Imatinib Cessation?

BCR-ABL1 transcripts at imatinib cessation were quantified by droplet digital PCR (ddPCR) for 175 patients on the STIM2 trial. Patients with BCR-ABL1 transcripts below a defined cutoff had a 12-month molecular recurrence rate of 46% versus 68% for those above the cutoff. Implications of using ddPCR in forecasting successful imatinib cessation are discussed.See related article by Nicolini et al., p. 6606.

Identification and Characterization of AES-135, a Hydroxamic Acid-Based HDAC Inhibitor That Prolongs Survival in an Orthotopic Mouse Model of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, incurable cancer with a 20% 1 year survival rate. While standard-of-care therapy can prolong life in a small fraction of cases, PDAC is inherently resistant to current treatments, and novel therapies are urgently required. Histone deacetylase (HDAC) inhibitors are effective in killing pancreatic cancer cells in in vitro PDAC studies, and although there are a few clinical studies investigating combination therapy including HDAC inhibitors, no HDAC drug or combination therapy with an HDAC drug has been approved for the treatment of PDAC. We developed an inhibitor of HDACs, AES-135, that exhibits nanomolar inhibitory activity against HDAC3, HDAC6, and HDAC11 in biochemical assays. In a three-dimensional coculture model, AES-135 kills low-passage patient-derived tumor spheroids selectively over surrounding cancer-associated fibroblasts and has excellent pharmacokinetic properties in vivo. In an orthotopic murine model of pancreatic cancer, AES-135 prolongs survival significantly, therefore representing a candidate for further preclinical testing.

Nuclear-Cytoplasmic Transport Is a Therapeutic Target in Myelofibrosis.

PURPOSE: Myelofibrosis is a hematopoietic stem cell neoplasm characterized by bone marrow reticulin fibrosis, extramedullary hematopoiesis, and frequent transformation to acute myeloid leukemia. Constitutive activation of JAK/STAT signaling through mutations in JAK2, CALR, or MPL is central to myelofibrosis pathogenesis. JAK inhibitors such as ruxolitinib reduce symptoms and improve quality of life, but are not curative and do not prevent leukemic transformation, defining a need to identify better therapeutic targets in myelofibrosis. EXPERIMENTAL DESIGN: A short hairpin RNA library screening was performed on JAK2V617F-mutant HEL cells. Nuclear-cytoplasmic transport (NCT) genes including RAN and RANBP2 were among top candidates. JAK2V617F-mutant cell lines, human primary myelofibrosis CD34+ cells, and a retroviral JAK2V617F-driven myeloproliferative neoplasms mouse model were used to determine the effects of inhibiting NCT with selective inhibitors of nuclear export compounds KPT-330 (selinexor) or KPT-8602 (eltanexor). RESULTS: JAK2V617F-mutant HEL, SET-2, and HEL cells resistant to JAK inhibition are exquisitely sensitive to RAN knockdown or pharmacologic inhibition by KPT-330 or KPT-8602. Inhibition of NCT selectively decreased viable cells and colony formation by myelofibrosis compared with cord blood CD34+ cells and enhanced ruxolitinib-mediated growth inhibition and apoptosis, both in newly diagnosed and ruxolitinib-exposed myelofibrosis cells. Inhibition of NCT in myelofibrosis CD34+ cells led to nuclear accumulation of p53. KPT-330 in combination with ruxolitinib-normalized white blood cells, hematocrit, spleen size, and architecture, and selectively reduced JAK2V617F-mutant cells in vivo. CONCLUSIONS: Our data implicate NCT as a potential therapeutic target in myelofibrosis and provide a rationale for clinical evaluation in ruxolitinib-exposed patients with myelofibrosis.

Similar expression profiles in CD34+ cells from chronic phase chronic myeloid leukemia patients with and without deep molecular responses to nilotinib.

The life expectancy of patients with chronic phase chronic myeloid leukemia on tyrosine kinase inhibitor therapy now approaches that of the general population. Approximately 60% of patients treated with second generation tyrosine kinase inhibitors achieve a deep molecular response, the prerequisite for a trial of treatment-free remission. Those patients unlikely to achieve deep molecular response may benefit from more intensive therapy up front. To identify biomarkers predicting deep molecular response we performed transcriptional profiling on CD34+ progenitor cells from newly diagnosed chronic phase chronic myeloid leukemia patients treated with nilotinib on a prospective clinical trial. Using unsupervised and targeted analytical strategies, we show that gene expression profiles are similar in patients with and without subsequent deep molecular response. This result is in contrast to the distinct expression signature of CD34+ chronic phase chronic myeloid leukemia patients failing to achieve a cytogenetic response on imatinib and suggests that deep molecular response to second-generation tyrosine kinase inhibitors is governed by the biology of more primitive chronic myeloid leukemia cells or extrinsic factors.

Autocrine Tnf signaling favors malignant cells in myelofibrosis in a Tnfr2-dependent fashion.

Tumor necrosis factor alpha (TNF) is increased in myelofibrosis (MF) and promotes survival of malignant over normal cells. The mechanisms altering TNF responsiveness in MF cells are unknown. We show that the proportion of marrow (BM) cells expressing TNF is increased in MF compared to controls, with the largest differential in primitive cells. Blockade of TNF receptor 2 (TNFR2), but not TNFR1, selectively inhibited colony formation by MF CD34+ and mouse JAK2V617F progenitor cells. Microarray of mouse MPN revealed reduced expression of X-linked inhibitor of apoptosis (Xiap) and mitogen-activated protein kinase 8 (Mapk8) in JAK2V617F relative to JAK2WT cells, which were normalized by TNFR2 but not TNFR1 blockade. XIAP and MAPK8 were also reduced in MF CD34+ cells compared to normal BM, and their ectopic expression induced apoptosis. Unlike XIAP, expression of cellular IAP (cIAP) protein was increased in MF CD34+ cells. Consistent with cIAP's role in NF-κB activation, TNF-induced NF-κB activity was higher in MF vs. normal BM CD34+ cells. This suggests that JAK2V617F reprograms TNF response toward survival by downregulating XIAP and MAPK8 through TNFR2. Our results reveal an unexpected pro-apoptotic role for XIAP in MF and identify TNFR2 as a key mediator of TNF-induced clonal expansion.

Minimal Residual Disease Eradication in CML: Does It Really Matter?

BCR-ABL1 tyrosine kinase inhibitors (TKIs) have improved the prognosis of chronic phase chronic myeloid leukemia (CP-CML) to an extent that survival is largely determined by non-CML mortality. Monitoring for minimal residual disease by measuring BCR-ABL1 messenger RNA is a key component of CML management. CP-CML patients who achieve a stable deep molecular response may discontinue (TKIs) with an ~ 50% chance of entering treatment-free remission (TFR). So far discontinuation of TKIs has largely been limited to clinical trials, but is on the verge of becoming a part of wider clinical practice. Careful patient selection, dense molecular monitoring, and prompt reinstitution of treatment in the event of relapse are all vital to reproduce the same level of success. Much effort has been dedicated to identifying therapeutic strategies to eliminate CML stem cells and enable to TFR in more patients. Unfortunately, despite promising preclinical data, as yet, none of the various approaches have entered clinical practice.

Mechanisms of Resistance to ABL Kinase Inhibition in Chronic Myeloid Leukemia and the Development of Next Generation ABL Kinase Inhibitors.

Chronic myeloid leukemia is increasingly viewed as a chronic illness; most patients have a life expectancy close to that of the general population. Despite progress made using BCR-ABL1 tyrosine kinase inhibitors (TKIs), drug resistance via BCR-ABL1-dependent and BCR-ABL1-independent mechanisms continues to be an issue. BCR-ABL1-dependent resistance is primarily mediated through oncoprotein kinase domain mutations and usually results in overt resistance to TKIs. However, BCR-ABL1-independent resistance in the setting of effective BCR-ABL1 inhibition is recognized as a major contributor to minimal residual disease. Efforts to eradicate persistent leukemic stem cells have focused on combination therapy.

Post-traumatic Stress and Trauma-Related Subjective Distress: Comparisons Among Hispanics, African-Americans, and Whites with Severe Mental Illness.

We tested the hypothesis with a sample of community mental health clients (N = 132) that Hispanic clients would report significantly greater post-traumatic stress symptoms than African-American or white clients when controlling for gender, psychiatric symptoms of SMI, and subjective distress from six of the most commonly reported trauma in the SMI literature. Results supported our main hypothesis: being self-identified as Hispanic was significantly associated with greater post-traumatic stress symptoms. Subjective distress from having been sexually abused along with being "Hispanic" were the only two significant variables left in the equation. Limitations of this study include its modest sample size.