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Thromboses are major causes of morbidity and mortality in polycythemia vera (PV) and essential thrombocythemia (ET) diseases associated with JAK2V617F mutation. However, the molecular mechanism(s) of increased thrombosis in PV and ET remain unknown. Kruppel-like factor 2 (KLF2) is a transcription factor that regulates expression of genes associated with inflammation and thrombosis; the absence of KLF2 in neutrophils causes thrombosis by inducing tissue factor. We studied the role of KLF2 in regulating prothrombotic gene expression in PV and ET. Neutrophils and platelets KLF2 expression in PV and ET was lower than the controls. Furthermore, in patients with thromboses, KLF2 transcripts were lower in platelets than those without thromboses. JAK2V617F allelic burden was inversely correlated with KLF2 transcript levels, suggesting JAK-STAT pathway may downregulate KLF2 expression. Whole transcriptome analyses of neutrophils and platelets showed that a lower KLF2 expression was associated with an upregulation of KLF2-regulated thrombotic genes. In addition, low KLF2 expression in platelets positively correlated with thrombotic events. In patients with PV and ET, KLF2 expression was induced by pegylated interferon alfa (PegINF-α) but not by hydroxyurea treatments. These data suggest that KLF2 may be a regulator of PV and ET thrombosis and a novel therapeutic target to prevent thrombosis.

Iron availability for erythropoiesis is controlled by the iron-regulatory hormone hepcidin. Increased erythropoiesis negatively regulates hepcidin synthesis by erythroferrone (ERFE), a hormone produced by erythroid precursors in response to erythropoietin (EPO). The mechanisms coordinating erythropoietic activity with iron homeostasis in erythrocytosis with low EPO are not well defined as exemplified by dominantly inherited (heterozygous) gain-of-function mutation of human EPO receptor (mtHEPOR) with low EPO characterized by postnatal erythrocytosis. We previously created a mouse model of this mtHEPOR that develops fetal erythrocytosis with a transient perinatal amelioration of erythrocytosis and its reappearance at 3-6 weeks of age. Prenatally and perinatally, mtHEPOR heterozygous and homozygous mice (differing in erythrocytosis severity) had increased Erfe transcripts, reduced hepcidin, and iron deficiency. Epo was transiently normal in the prenatal life; then decreased at postnatal day 7, and remained reduced in adulthood. Postnatally, hepcidin increased in mtHEPOR heterozygotes and homozygotes, accompanied by low Erfe induction and iron accumulation. With aging, the old, especially mtHEPOR homozygotes had a decline of erythropoiesis, myeloid expansion, and local bone marrow inflammatory stress. In addition, mtHEPOR erythrocytes had a reduced lifespan. This, together with reduced iron demand for erythropoiesis, due to its age-related attenuation, likely contributes to increased iron deposition in the aged mtHEPOR mice. In conclusion, the erythroid drive-mediated inhibition of hepcidin production in mtHEPOR mice in the prenatal/perinatal period is postnatally abrogated by increasing iron stores promoting hepcidin synthesis. The differences observed in studied characteristics between mtHEPOR heterozygotes and homozygotes suggest dose-dependent alterations of downstream EPOR stimulation.

Hotspots difficult to confidently assess for CHIP

Supplementary Methods and Supplementary Figures

Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by detectable hematopoietic-associated gene mutations in a person without evidence of hematologic malignancy. We sought to identify additional cancer-presenting mutations usable for CHIP detection by performing a data mining analysis of 48 somatic mutation landscape studies reporting mutations at diagnoses of 7,430 adult and pediatric patients with leukemia or other hematologic malignancy. Following extraction of 20,141 protein-altering mutations, we identified 434 significantly recurrent mutation hotspots, 364 of which occurred at loci confidently assessable for CHIP. We then performed an additional large-scale analysis of whole-exome sequencing data from 4,538 persons belonging to three noncancer cohorts for clonal mutations. We found the combined cohort prevalence of CHIP with mutations identical to those reported at blood cancer mutation hotspots to be 1.8%, and that some of these CHIP mutations occurred in children. Our findings may help to improve CHIP detection and precancer surveillance for both children and adults.Significance:This study identifies frequently occurring mutations across several blood cancers that may drive hematologic malignancies and signal increased risk for cancer when detected in healthy persons. We find clonal mutations at these hotspots in a substantial number of individuals from noncancer cohorts, including children, showcasing potential for improved precancer surveillance.See related commentary by Spitzer and Levine, p. 192.

Introduction : Treatment options in patients with myelofibrosis (MF) presenting with thrombocytopenia are limited. Final results of the phase 2 study (NCT01348490) of ruxolitinib in patients with MF and low baseline platelet counts (50–100 × 109/L) are reported. Patients and Methods : Patients received ruxolitinib 5 mg twice daily (BID), with optional up-titration to a maximum of 15 mg BID, provided platelet count remained ≥40 × 109/L. Assessments included spleen volume and length, Total Symptom Score (TSS), quality of life, and safety. Results : Of 66 patients, 52 (78.8%) completed the first 24 weeks of treatment. Median (range) percentage change from baseline in spleen volume and TSS (coprimary endpoints) were −20.5% (−55.8% to 38.5%, n=51) and −39.8% (−98.6% to 226.4%, n=53), respectively; greatest median reductions were in the 10 mg BID final titrated dose group. Of patients achieving ≥35% or ≥10% reduction in spleen volume, 8/11 (72.7%) and 21/34 (61.8%), respectively, were in the 10 mg BID final titrated dose group. Thirty-seven of 65 patients (56.9%) had ≥20% improvement in TSS, and 35/66 patients (53.0%) were Patient Global Impression of Change responders. Treatment-emergent adverse events led to dose interruption in 17/66 patients (25.8%), most commonly thrombocytopenia (n=3). Conclusion : A starting dose of ruxolitinib 5 mg BID with gradual up-titration and dose optimization based on hematologic parameters and response was efficacious and generally well-tolerated in patients with MF and low platelet counts. Median improvement in spleen volume and symptoms was greatest for patients receiving ruxolitinib 10 mg BID.

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Supplementary Methods, Legends

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Supplementary Table 1

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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.

Supplementary Table 2

Supplementary Table 3-8

Three well-studied populations living at high altitudes are Tibetans, Andeans (Aymaras and Quechuas), and Ethiopians. Unlike Tibetans and Ethiopians who have similar hemoglobin (Hb) levels as individuals living at sea level, Aymara Hb levels increase when living at higher altitudes. Our previous whole genome study of Aymara people revealed several selected genes that are involved in cardiovascular functions, but their relationship with Hb levels was not elucidated. Here, we studied the frequencies of known evolutionary-selected variants in Tibetan and Aymara populations and their correlation with high Hb levels in Aymara. We genotyped 177 Aymaras at three different altitudes: 400 m (Santa Cruz), 4000 m (La Paz), and 5000 m (Chorolque), and correlated the results with the elevation of residence. Some of the Tibetan-selected variants also exist in Aymaras, but at a lower prevalence. Two of 10 Tibetan selected variants of EPAS1 were found (rs13005507 and rs142764723) and these variants did not correlate with Hb levels. Allele frequencies of 5 Aymara selected SNPs (heterozygous and homozygous) at 4000 m (rs11578671_BRINP3, rs34913965_NOS2, rs12448902_SH2B1, rs10744822_TBX5, and rs487105_PYGM) were higher compared to Europeans. The allelic frequencies of rs11578671_BRINP3, rs34913965_NOS2, and rs10744822_SH2B1 were significantly higher for Aymaras living at 5000 m than those at 400 m elevation. Variant rs11578671, close to the BRINP3 coding region, correlated with Hb levels in females. Variant rs34913965 (NOS2) correlated with leukocyte counts. Variants rs12448902 (SH2B1) and rs34913965 (NOS2) associated with higher platelet levels. The correlation of these SNPs with blood cell counts demonstrates that the selected genetic variants in Aymara influence hematopoiesis and cardiovascular effects.

Background Myelofibrosis (MF) is a myeloproliferative neoplasm that commonly harbors acquired somatic gene mutations in JAK2, CALR, or MPL. In the Phase 3 COMFORT-1 trial, which enrolled 309 patients (pts) with JAK inhibitor-naïve MF, ruxolitinib (RUX) showed spleen volume reduction of ≥35% (SVR35) and an improvement of 50% or more in the total symptom score (TSS50) in 42% and 46%, respectively compared to placebo. Activity has been shown with the combination of selinexor (SEL) plus RUX in preclinical studies. Methods XPORT-MF-034 is an open-label, Phase 1/2 study (NCT04562389) to evaluate the safety and efficacy of SEL plus RUX in treatment-naïve MF pts. SEL is evaluated at 2 dose levels, 40mg and 60mg once-weekly plus twice daily RUX in 28-day cycles. For nausea, all pts receive prophylaxis with a 5-HT3 antagonist prior to each SEL dose and as needed. Primary endpoints are to determine maximum tolerated dose, recommended Phase 2 dose (RP2D), and safety. Secondary endpoints include spleen and symptom response, and hemoglobin stabilization and improvement. The efficacy population for spleen and symptom evaluable pts included those who had a spleen assessment or at least one symptom score available, respectively, at baseline and the W12 or W24 timepoint. Results As of Oct 21, 2022, 24 pts have received at least one dose of 40mg or 60mg weekly SEL with RUX twice daily as per standard of care. Median age was 64 years old (range 44-77) and 11 pts had primary MF, 6 had post-ET MF, and 7 had post-PV MF. DIPSS risk category was int-1, int-2, and high risk for 7, 11, and 6, respectively. The median daily dose of ruxolitinib received was 20 mg. In efficacy evaluable pts, 63% (12/19) and 92% (11/12) achieved SVR35 at W12 and W24, and 83% (10/12) and 67% (4/6) achieved TSS50 at W12 and W24. Among the 11 pts who had a baseline hemoglobin level Conclusions To date, in pts with treatment-naïve MF, the novel combination of SEL and RUX has been reasonably well-tolerated with a generally manageable safety profile and has shown encouraging activity in spleen and symptom responses, in addition to hemoglobin stabilization. Updated safety and efficacy, including symptom data amongst those pts non evaluable for TSS50 at the time of the Oct data cutoff, as well as RP2D, will be available for presentation at AACR 2023. Citation Format: Haris Ali, Ashwin Kishtagari, Keri Maher, Sanjay Mohan, Karen Ansaldo, Xulong Wang, Kamal Chamoun, Josef T. Prchal, Srinivas K. Tantravahi. A Phase 1, open-label, dose-escalation study of selinexor plus ruxolitinib in patients with treatment-naïve myelofibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT261.

To compare total serum bilirubin (TSB) levels, phototherapy usage, and hospital readmission for jaundice among neonates with Down syndrome vs controls.A retrospective cohort study using 15 years of multihospital data. We created control reference intervals (5th, median, and 95th percentiles) for initial TSB values hourly during the first days after birth, and determined the proportion of neonates with Down syndrome whose TSB exceeded the 95th percentile control interval. We determined the proportion with an initial TSB exceeding the upper control reference interval, the highest TSB recorded, the percentage of neonates receiving phototherapy, and the rate of hospital readmission for jaundice treatment.We compared 357 neonates with Down syndrome with 377 368 controls. Compared with controls, those with Down syndrome had 4.7 times the risk (95% CI, 3.9-5.7; P .0001) of an initial TSB exceeding the 95th percentile control interval (23.5% vs 5.0%), 8.9 times (95% CI, 8.1-9.8; P .0001) the phototherapy usage (62.2% vs 7.0%), and 3.6 times (95% CI, 1.6-8.2; P = .0075) the readmission rate for jaundice (17.4 vs 4.8 per 1000 live births).Neonates with Down syndrome have a substantial risk of early hyperbilirubinemia. The American Academy of Pediatrics currently advises obtaining an early screening complete blood count from neonates with Down syndrome. We submit that assessing their TSB is also advisable.

We report a novel glucose-6-phosphate dehydrogenase (G6PD) variant (c.1375C>G) discovered in a 3-day-old Hispanic male child from Salt Lake City, UT, USA. This newborn presented with severe hyperbilirubinemia (29.8 mg/dL or 510 μmol/L) and marked hemolysis evidenced by elevated end-tidal carbon monoxide concentration (5.9 ppm, normal G6PD variant. His mother was heterozygous for this same variant, and she had a moderate decrease in G6PD enzyme activity (7.1 U/g Hb). On the basis of these findings, we propose this variant as a novel pathogenic mutation.

Portal hypertension (pHTN) complicates myeloproliferative neoplasms (MPNs), and usually occurs due to Budd-Chiari syndrome or splanchnic vein thrombosis. Current management modalities for MPN-associated pHTN include anticoagulation, transjugular intrahepatic portosystemic shunt (TIPS), and orthotopic liver transplant. Data on the thrombotic and bleeding outcomes of this practice is of poor quality, and whether direct oral anticoagulants (DOACs) are effective in this setting is unknown. We describe failure of DOACs to prevent post-TIPS complications in two case reports of patients with MPN-associated pHTN and review the associated literature. We conducted a comprehensive search in Embase (embase.com), Scopus (scopus.org), and PubMed for existing data on MPN-associated pHTN post-TIPS procedure. Four studies (n = 251) of patients with pHTN post-TIPS were eligible (MPN, n = 143). A review of the literature suggests that patients with MPN-associated pHTN may be at higher risk for post-TIPS complications including stent thrombosis and stenosis, compared with other causes of thrombotic pHTN. DOAC use has not been studied in this setting. While further studies to guide optimal management of MPN-associated pHTN post-TIPS are needed, available evidence suggests that life-long anticoagulation is warranted. DOACs should not be considered standard of care because of lack of evidence of efficacy.

Methemoglobinemia is a rare disorder associated with oxidization of divalent ferro-iron of hemoglobin (Hb) to ferri-iron of methemoglobin (MetHb). Methemoglobinemia can result from either inherited or acquired processes. Acquired forms are the most common, mainly due to the exposure to substances that cause oxidation of the Hb both directly or indirectly. Inherited forms are due either to autosomal recessive variants in the CYB5R3 gene or to autosomal dominant variants in the globin genes, collectively known as HbM disease. Our recommendations are based on a systematic literature search. A series of questions regarding the key signs and symptoms, the methods for diagnosis, the clinical management in neonatal/childhood/adulthood period, and the therapeutic approach of methemoglobinemia were formulated and the relative recommendations were produced. An agreement was obtained using a Delphi-like approach and the experts panel reached a final consensus >75% of agreement for all the questions.

Introduction Essential thrombocythemia (ET) and polycythemia vera (PV) patients suffer from various symptoms that worsen quality of life (QOL), yet serial data on symptom changes resulting from therapy are sparse in the literature. Patient questionnaires from 2 large multicenter trials (MPN-RC 111, 112) were used to assess change in symptom burden and QOL over 12 months and impact of baseline symptom burden on subsequent change in ET / PV patients on hydroxyurea (HU) or pegylated interferon alfa-2a (PEG). Methods Trials MPN-RC 111 was a single-arm, open-label, phase II trial evaluating response to PEG in high-risk ET / PV patients with HU resistance/intolerance or splanchnic vein thrombosis (SVT; NCT01259817). MPN-RC 112 was a randomized, open-label, phase III trial comparing response to PEG versus HU in cytoreductive therapy naïve high-risk ET / PV patients diagnosed < 5 years ago (NCT01258856). Measures Patients reported disease-related symptoms via the validated Myeloproliferative Neoplasms Symptom Assessment Form (MPN-SAF), QOL via the European Organisation for the Research and Treatment of Cancer Core QOL Questionnaire (EORTC QLQ-C30), and (if applicable) PEG-related symptoms (flu-like symptoms, injection site irritation, blurry vision, vision change, flushing) at baseline, 3, 6, 9, and 12 months. Analysis Mixed models assessed mean changes from baseline in the MPN-SAF Total Symptom Score (TSS), MPN-SAF items, QOL, and PEG-related symptoms in MPN-RC 111, 112 PEG, and 112 HU patients. Mixed models also assessed the impact of baseline symptom burden (high [TSS ≥ 20] versus low) on subsequent change in PEG (MPN-RC 111 and 112) and HU patients. Results Patients Of the 135 enrolled MPN-RC 111 patients, 20 with SVT and 1 with no questionnaires were excluded. Of the remaining 114, 64 (56%) / 50 (44%) had ET / PV. Patients were 51% / 48% female. Median age was 65 / 64 years, and median time since diagnosis was 38 / 55 months. 31% / 22% had prior thrombosis, and 19% / 56% had splenomegaly. Of the 168 enrolled MPN-RC 112 patients (82 PEG, 86 HU), 2 with no questionnaires were excluded. Of the remaining 166, 79 (48%) / 87 (52%) had ET / PV. Patients were 50% / 33% female. Median age was 60 / 62 years, and median time since diagnosis was 3 / 3 months. 25% / 29% had prior thrombosis, and 11% / 37% had splenomegaly. Symptoms Questionnaire completion rates ranged from 90 - 99%, 87 - 100%, and 75 - 96% for on-treatment MPN-RC 111, 112 PEG, and 112 HU patients. At baseline, TSS (0 [absent] - 100 [worst imaginable]) and QOL (0 [very poor] - 100 [excellent]) means (SDs) were 19.5 (18.4) and 71.6 (20.1) for MPN-RC 111, 17.0 (13.6) and 67.9 (24.3) for MPN-RC 112 PEG, and 14.6 (11.4) and 73.8 (18.8) for MPN-RC 112 HU patients. On average, MPN-RC 111 patients had significant improvement of TSS, fatigue, abdominal pain, abdominal discomfort, dizziness, numbness, night sweats, and fever; MPN-RC 112 PEG patients had significant worsening of fever; and MPN-RC 112 HU patients had significant worsening of inactivity (no mean changes indicating improvement were observed). PEG patients had significant worsening of PEG-related symptoms. The greatest improvements occurred in the 46 (40%), 27 (33%), and 23 (28%) MPN-RC 111, 112 PEG, and 112 HU patients with high baseline symptom burden. On average, PEG patients with high baseline symptom burden had significant improvement of TSS, fatigue, early satiety, abdominal pain, abdominal discomfort, inactivity, headache, concentration, dizziness, numbness, insomnia, cough, night sweats, itching, bone pain, fever, weight loss, and QOL, while those with low baseline symptom burden had significant worsening of TSS, early satiety, headache, itching, and bone pain. On average, HU patients with high baseline symptom burden had significant improvement of TSS, early satiety, abdominal discomfort, headache, dizziness, numbness, insomnia, itching, and weight loss, while those with low baseline symptom burden had significant worsening of TSS, early satiety, abdominal discomfort, inactivity, concentration, and sexual desire/function (Figures 1 and 2). Conclusions Although no statistical comparisons were made across trials, overall improvements were seen in MPN-RC 111 but not 112. Patients with high baseline symptom burden experienced the greatest improvements in symptom burden and QOL during treatment with PEG or HU, which may explain the improvements seen in the more advanced patients in MPN-RC 111 compared to 112. Disclosures Mascarenhas: Celgene, Prelude, Galecto, Promedior, Geron, Constellation, and Incyte: Consultancy; Incyte, Kartos, Roche, Promedior, Merck, Merus, Arog, CTI Biopharma, Janssen, and PharmaEssentia: Other: Research funding (institution). Yacoub:Dynavax: Current equity holder in publicly-traded company; Ardelyx: Current equity holder in publicly-traded company; Cara Therapeutics: Current equity holder in publicly-traded company; Hylapharm: Current equity holder in private company; Incyte: Speakers Bureau; Agios: Honoraria, Speakers Bureau; Novartis: Speakers Bureau; Roche: Other: Support of parent study and funding of editorial support. Hoffman:Protagonist: Consultancy; Forbius: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Dompe: Research Funding. Silver:PharmaEssentia: Speakers Bureau. Mesa:Bristol Myers Squibb: Research Funding; Incyte: Research Funding; AbbVie: Research Funding; Samus Therapeutics: Research Funding; Genentech: Research Funding; CTI BioPharma: Research Funding; Promedior: Research Funding; Sierra Oncology: Consultancy; LaJolla Pharmaceutical Company: Consultancy; Novartis: Consultancy.

The patients with mantle cell lymphoma (MCL) have translocation t(11;14) associated with cyclin D1 overexpression. We observed that iron (an essential cofactor of dioxygenases including prolyl hydroxylases [PHDs]) depletion by deferoxamine blocked MCL cells’ proliferation, increased expression of DNA damage marker γH2AX, induced cell cycle arrest and decreased cyclin D1 level. Treatment of MCL cell lines with dimethyloxalylglycine, which blocks dioxygenases involving PHDs by competing with their substrate 2‐oxoglutarate, leads to their decreased proliferation and the decrease of cyclin D1 level. We then postulated that loss of EGLN2/PHD1 in MCL cells may lead to down‐regulation of cyclin D1 by blocking the degradation of FOXO3A, a cyclin D1 suppressor. However, the CRISPR/Cas9‐based loss‐of‐function of EGLN2/PHD1 did not affect cyclin D1 expression and the loss of FOXO3A did not restore cyclin D1 levels after iron chelation. These data suggest that expression of cyclin D1 in MCL is not controlled by ENGL2/PHD1‐FOXO3A pathway and that chelation‐ and 2‐oxoglutarate competition‐mediated down‐regulation of cyclin D1 in MCL cells is driven by yet unknown mechanism involving iron‐ and 2‐oxoglutarate‐dependent dioxygenases other than PHD1. These data support further exploration of the use of iron chelation and 2‐oxoglutarate‐dependent dioxygenase inhibitors as a novel therapy of MCL.

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.