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.
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 K562R, 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 K562R 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-ABL1T315I 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.
•A function-first shRNA library screen identifies pathways involved in BCR-ABL1 kinase-independent TKI resistance.•RAN or XPO1 inhibition impairs survival of progenitors from newly diagnosed or TKI-resistant CML patients.
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
, or
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.
A short hairpin RNA library screening was performed on JAK2
-mutant HEL cells. Nuclear-cytoplasmic transport (NCT) genes including
and
were among top candidates. JAK2
-mutant cell lines, human primary myelofibrosis CD34
cells, and a retroviral JAK2
-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).
JAK2
-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 JAK2
-mutant cells
.
Our data implicate NCT as a potential therapeutic target in myelofibrosis and provide a rationale for clinical evaluation in ruxolitinib-exposed patients with myelofibrosis.
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.
Abstract
Standard of care for AML includes chemotherapy and stem cell transplant, with 5-year survival rates <30%. We sought to identify genes critical to AML cells, irrespective of mutational ...status, and performed an shRNA screen targeting 1,287 genes on 12 AML patient samples. This screen identified Sirtuin 5 (SIRT5) as a top candidate. SIRT5 is the only known enzyme with desuccinylase, demalonylase, and/or deglutarylase activity and we are the first to report the dependence of AML cells on SIRT5. Next, we stably transduced a panel of AML cell lines with doxycycline (dox)-inducible shSIRT5 (dox-shSIRT5). SIRT5 knockdown (KD) strongly inhibited cell growth, colony formation and increased apoptosis in 15/22 lines (SIRT5-dependent), while 7/22 lines were SIRT5-independent. SIRT5 dependence did not correlate with AML-related mutations nor basal SIRT5 expression. SIRT5 KD in primary AML samples (N=25) revealed a therapeutic window (~50% reduction), with no effect in CB samples (N=5). We examined the requirement of SIRT5 in vivo using three mouse models of leukemia. In a xenograft model with AML cell lines, SIRT5 KD indefinitely prolonged survival of mice injected with SIRT5-dependent cells with no sign of leukemia. Bone marrow transplant with transduced (MLL-AF9 or BCR-ABL1) SIRT5 null cells showed reduced leukemia cell burden and splenomegaly, and significantly prolonged survival. FLT3-ITD-driven disease was also blunted by the absence of SIRT5 in a genetic knockout mouse model. Mechanically, SIRT5 KD profoundly reduced oxidative phosphorylation (OXPHOS) and glycolysis. Additionally, SIRT5 KD increased mitochondrial superoxide selectively in annexin V-negative, SIRT5-dependent cells. Concomitant, ectopic expression of SOD2 abrogated the increase in superoxide, rescued cells from apoptosis, and rescued the colony formation deficit. Untargeted metabolomics revealed RNA charging and alanine and serine metabolism as top metabolic pathways regulated by SIRT5, with glutaminase (GLS) and α-ketoglutarate identified as potential upstream regulators. Metabolic tracing experiments with 13C5,15N2-glutamine confirmed disrupted glutamine metabolism in SIRT5-dependent cells. Together, these results indicate that SIRT5 is required to regulate glutamine flux to sustain redox homeostasis and/or anabolism. NRD167, a novel SIRT5 inhibitor, was used to target SIRT5 in AML. NRD167 reduced cell proliferation, induced apoptosis, and reduced OXPHOS in SIRT5-dependent but not SIRT5-independent cells. NRD167 inhibited colony formation from AML patient samples, but not in CB samples. An AML patient-derived xenograft model trended toward prolonged survival following ex vivo treatment with NRD167. Our data suggest that the majority of AML samples are dependent on SIRT5 and that inhibition preferentially targets AML cells, implicating SIRT5 as a therapy target in AML.
Citation Format: Dongqing Yan, Anca Franzini, Anthony D. Pomicter, Brayden J. Halverson, Orlando Antelope, Clinton C. Mason, Jonathan M. Ahmann, Anna V. Senina, Courtney L. L. Jones, Matthew S. Zabriskie, Hein Than, Michael J. Xiao, Alexandria van Scoyk, Ami B. Patel, William L. L. Heaton, Shawn C. Owen, Joshua L. Andersen, Christina M. Egbert, Julie A. Reisz, Angelo D'Alessandro, James E. Cox, Kevin C. Gantz, Hannah M. Redwine, Siddharth M. Iyer, Jamshid S. Khorashad, Nima Rajabi, Christian A. Olsen, Thomas O'Hare, Michael W. Deininger. A critical role for SIRT5 in acute myeloid leukemia metabolism 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 LB109.
▪
Myelofibrosis (MF) is a hematopoietic stem cell neoplasm characterized by constitutive activation of JAK/STAT signaling due to mutations in JAK2, calreticulin or MPL. Many MF patients suffer from ...severe constitutional symptoms and have reduced life expectancy due to cytopenias, progression to acute myeloid leukemia and thromboembolic events. JAK kinase inhibitors such as ruxolitinib (RUX) reduce MF symptoms, but like all other drugs used in MF, are not curative, with persistence of mutant cells and prompt symptom rebound upon discontinuation. This defines a clinical need to identify strategies capable of inducing more profound and durable responses in MF.
To identify previously unrecognized molecular vulnerabilities in MF, we infected HEL cells (homozygous for JAK2V617F) with a barcoded lentiviral shRNA library targeting ~5,000 human signal transduction genes, with 5-6 shRNAs/gene (Cellecta Human Module 1). Conditions were optimized to achieve a multitude of infection (MOI) of ~1. Barcode abundance was compared between days 0 and 9 after infection by next generation sequencing. Candidates were selected based on ≥ 15-fold reduction of abundance by ≥ 2 shRNAs targeting the same gene, similar to Khorashad et al. Blood. 2015;125(11):1772-81. Amongst the genes meeting these criteria, nuclear cytoplasmic transport (NCT) was significantly enriched, with RAN and RANBP2 amongst the top genes, suggesting that HEL cells may be highly dependent on NCT. For confirmation, HEL cells were stably transduced with doxycycline (DOX)-inducible shRAN. After 72 hours DOX-induced knockdown of RAN reduced viable cells by 77.3±5.5% and colony formation by 82.8±1.3% and dramatically increased apoptosis (uninduced: ~10% vs. induced: ~50%). Similar results were observed in SET-2 cells (heterozygous for JAK2V617F). We next cultured HEL and SET-2 cells with graded concentrations of the KPT-330 (selinexor, Karyopharm), an inhibitor of CRM-1, the core component of NCT, or RUX as a comparison. Selinexor was five-fold more potent than RUX against HEL cells (IC50: 98nM for KPT vs. 536 nM for RUX) and as potent as RUX in SET-2 cells (IC50:~100 nM). Importantly, RUX-resistant HEL cells (IC50:24µM) were highly sensitive to inhibition of NCT by knockdown of RAN or selinexor (IC50:160nM). Selinexor also selectively inhibited colony formation by primary MF vs. cord blood (CB) CD34+ cells (IC50:93nM for MF vs. 203nM for CB). Lastly, selinexor enhanced RUX-induced growth inhibition and apoptosis in primary MF CD34+cells cultured ex vivo for 72h (including both JAK2 mutation positive and negative MF samples, n=3 for each, and RUX resistant patient samples, n=6). Nuclear:cytoplasmic fractionation of HEL cells revealed that the expression and nuclear localization of the tumor suppressors FoxO3A and APC, but not of PP2A and nucleophosmin (NPM) were significantly increased upon knockdown of RAN, which may contribute to the increased apoptosis following NCT inhibition.
To determine the in vivo effects of selinexor in MF, we induced MPN in Balb/c mice by transplanting donor marrow infected with JAK2V617F for three weeks, and then treated mice (n=13/group) with vehicle, selinexor (initial dose 20 mg/kg, 3x weekly, orally) or RUX (initial dose 50 mg/kg twice daily, orally) or the combination of RUX plus selinexor for up to 4 weeks. Combination treatment significantly reduced white blood cell counts and normalized spleen size. Compared to vehicle, selinexor alone significantly reduced GFP+cells in the spleen, and this effect was further enhanced with the combination treatment. Histopathology revealed that combination treatment restored splenic architecture, while bone marrow fibrosis was not significantly altered by selinexor or the combination. Mice in all groups, including the combined vehicle controls, experienced considerable weight loss, suggesting that toxicity may be partially due to high dose and frequent drug administration. Experiments with the next generation NCT inhibitor KPT-8602 Etchin et al., Leukemia, 2016 Jun 24 are underway.
In summary, our results suggest that MF cells are exquisitely dependent on NCT, and that NCT inhibition alone or in combination with RUX may reduce JAK2V617F allelic burden. This identifies NCT as a prime therapeutic target in MF. A phase I clinical trial of selinexor in refractory MF is in preparation.
Baloglu:Karyopharm Therapeutics: Employment, Equity Ownership. Deininger:BMS: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.
Introduction. Development of abnormal Philadelphia (Ph) negative clones following treatment of chronic myeloid leukemia (CML) patients with imatinib has been observed in 3 to 9% of patients. Here we ...report on a 77 year old male diagnosed with CML that responded to imatinib treatment and subsequently developed chronic myelomonocytic leukemia (CMML). He achieved major cytogenetic response within 3 months but this response coincided with the emergence of monocytosis diagnosed as CMML. Five months after starting imatinib treatment the patient succumbed to CMML. We analyzed five sequential samples to determine whether a chronological order of mutations defined the emergence of CMML and to characterize the clonal evolution of the CMML population.
Materials and Method. Five samples (diagnostic and four follow up samples) were available for analysis. CMML mutations were identified by whole exome sequencing (WES) in CD14+ cells following the onset of CMML, using CD3+ cells as constitutional control. Mutations were validated by Sequenom MassARRAY and Sanger sequencing and quantified by pyrosequencing. Deep WES was performed on the diagnostic sample to determine whether the mutations were present at CML diagnosis. To determine the clonal architecture of the emerging CMML, colony formation assays were performed on the diagnostic and the next two follow-up samples (Samples 1-3). More than 100 colonies per sample were plucked for DNA and RNA isolation. The DNA from these colonies were tested for the presence of the confirmed CMML mutations and the RNA was used for detection of BCR-ABL1 transcript using a Taqman real time assay.
Results. Four mutations were identified by Sequenom and WES throughout the patient's time course KRASG12R, MSLNP462H, NTRK3V443I and EZH2I669M . Sequenom did not identify these at diagnosis while deep WES did. Clones derived from colony formation assay revealed three distinct clones present in all samples analysed. Clone 1 had only KRASG12R, clone 2 had KRASG12R, MSLNP462H, and NTRK3V443I, and clone 3 had all four mutations. All clones containing any of these four mutations were BCR/ABL1 negative. Analysis of clonal architecture indicated that KRASG12R was acquired first and EZH2I669M last, while MSLNP462H and NTRK3V443I were acquired in between. These CMML clones increased proportionately as clinical CML metamorphosed into clinical CMML after initiation of imatinib therapy. Consistent with the colony data, pyrosequencing revealed that the ratio between the mutants remained largely stable throughout the follow up period.
Conclusion. This case illustrates how targeted therapy impacts clonal competition in a heterogeneous MPN. While the CML clone was dominant in the absence of imatinib, it was quickly outcompeted by the CMML clones upon initiation of imatinib therapy. The clonal architecture analysis, in combination with in vivo kinetics data, suggest that the KRASG12R mutation alone was able to produce a CMML phenotype as clones with just KRASG12R remained at a relatively stable ratio during follow up. Unexpectedly, acquisition of additional mutations, including EZH2I669M as the last mutational event identified in this patient, did not increase clonal competitiveness, at least in the peripheral blood. These data show that clonal evolution may not invariably increase clonal fitness, suggesting that factors other than Darwinian pressures contribute to clonal diversity in myeloproliferative neoplasms.
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Deininger:Gilead: Research Funding; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.
Current diagnostic criteria for bronchopulmonary dysplasia rely heavily on the level and duration of oxygen therapy, do not reflect contemporary neonatal care, and do not adequately predict childhood ...morbidity.
To determine which of 18 prespecified, revised definitions of bronchopulmonary dysplasia that variably define disease severity according to the level of respiratory support and supplemental oxygen administered at 36 weeks' postmenstrual age best predicts death or serious respiratory morbidity through 18-26 months' corrected age.
We assessed infants born at less than 32 weeks of gestation between 2011 and 2015 at 18 centers of the Eunice Kennedy Shriver National Institute of Child Health and Human Development Neonatal Research Network.
Of 2,677 infants, 683 (26%) died or developed serious respiratory morbidity. The diagnostic criteria that best predicted this outcome defined bronchopulmonary dysplasia according to treatment with the following support at 36 weeks' postmenstrual age, regardless of prior or current oxygen therapy: no bronchopulmonary dysplasia, no support (
= 773); grade 1, nasal cannula ≤2 L/min (
= 1,038); grade 2, nasal cannula >2 L/min or noninvasive positive airway pressure (
= 617); and grade 3, invasive mechanical ventilation (
= 249). These criteria correctly predicted death or serious respiratory morbidity in 81% of study infants. Rates of this outcome increased stepwise from 10% among infants without bronchopulmonary dysplasia to 77% among those with grade 3 disease. A similar gradient (33-79%) was observed for death or neurodevelopmental impairment.
The definition of bronchopulmonary dysplasia that best predicted early childhood morbidity categorized disease severity according to the mode of respiratory support administered at 36 weeks' postmenstrual age, regardless of supplemental oxygen use.
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