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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.

Standard of care for AML includes chemotherapy and stem cell transplant, with 5-year survival rates 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.

Background: There is growing interest in the application of palliative care principles to improve care for patients and families affected by neurologic diseases. We developed an interdisciplinary outpatient clinic for patients and families affected by neurologic disorders to better address the problems faced by our highest need patients. We have developed and improved this program over the past three years and share several of our most important lessons as well as ongoing challenges and areas where we see our clinic evolving in the future. Methods: We provide a description of our clinic logistics, including key steps in the initiation of the clinic, and provide descriptions from similar clinics at other institutions to demonstrate some of the variability in this growing field. We also provide results from a formal one-year quality improvement project and a one-year retrospective study of patients attending this clinic. Results: Our clinic has grown steadily since its inception and maintains high satisfaction ratings from patients, caregivers, and referring providers. To maintain standardized and efficient care we have developed materials for patients and referring physicians as well as checklists and other processes used by our interdisciplinary team. Feedback from our quality improvement project helped define optimal visit duration and refine communication among team members and with patients and families. Results from our chart review suggest our clinic influences advance care planning and place of death. Common referral reasons include psychosocial support, complex symptom management, and advance care planning. Current challenges for our clinic include developing a strategy for continued growth, creating a sustainable financial model for interdisciplinary care, integrating our services with disease-specific sections, improving primary palliative care knowledge and skills within our referral base, and building effective alliances with community neurologists, geriatrics, primary care, nursing homes, and hospices. Conclusions: Specialized outpatient palliative care for neurologic disorders fills several important gaps in care for this patient population, provides important educational opportunities for trainees, and creates opportunities for patient and caregiver-centered research. Educational initiatives are needed to train general neurologists in primary palliative care, to train neurologists in specialist palliative care, and to train palliative medicine specialists in neurology. Research is needed to build an evidence base to identify patient and caregiver needs, support specific interventions, and to build more efficient models of care in both academic and community settings.

Summary Objective Examine therapeutic potential of a selective serotonin reuptake inhibitor (SSRI) and a norepinephrine reuptake inhibitor (NERI) in an animal model of comorbidity between epilepsy, depression-like, and impulsive-like impairments. Methods Epilepsy was induced in male Wistar rats by LiCl and pilocarpine. An SSRI fluoxetine (FLX), and an NERI reboxetine (RBX) were administered either alone or as a combination over 1 week. Depressive-like and impulsive-like behaviors were examined using the forced swim test. Fast scan cyclic voltammetry was used to analyze serotonergic transmission in the raphe nucleus (RN)–prefrontal cortex (PFC) pathway, and noradrenergic transmission in locus coeruleus (LC)-PFC, and LC-RN projections. Monoamine levels in PFC were measured using high-performance liquid chromatography (HPLC). Functional capacities of 5-HT1A receptors and α2A adrenoreceptors in PFC were analyzed by autoradiography. Results Epileptic rats showed behavioral signs of depression and hyperimpulsivity, suppressed serotonergic and noradrenergic tones, decreased levels of serotonin (5-HT), and norepinephrine (NE); 5-HT1A receptor and α2A adrenoreceptors functions remained intact. FLX failed to improve behavioral deficits, but effectively raised 5-HT level and marginally improved RN-PFC serotonergic transmission. RBX reversed impulsive-like behavior, normalized content of NE and noradrenergic tone in LC-PFC and LC-RN. FLX-RBX combination fully reversed depressive-like behavior, and normalized RN-PFC serotonergic transmission. None of the treatment modified the function of 5-HT and NE receptors. Significance Depressive- and impulsive-like behaviors in the pilocarpine model of epilepsy stem respectively from dysfunctions of serotonergic and noradrenergic ascending pathways. At the same time, epilepsy-associated depression is SSRI resistant. The finding that an SSRI-NERI combination exerts antidepressant effect, along with RBX-induced improvement of LC-RN noradrenergic transmission point toward the involvement of LC-RN noradrenergic input in enabling therapeutic potential of FLX. Medications that improve serotonergic and noradrenergic transmission, such as serotonin–norepinephrine reuptake inhibitors may be effective in treating epilepsy-associated SSRI-resistant depression, as well as concurrent depression and attention-deficit/hyperactivity disorder (ADHD).

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. Disclosures 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.

Background: We have previously demonstrated that the transcriptional profile of diagnostic CD34+ cells from chronic phase chronic myeloid leukemia (CP-CML) patients exhibiting primary cytogenetic resistance to imatinib overlaps with that of patients with myeloid blast phase CML (BP-CML) (McWeeney et al. Blood 2010). These data suggest that primary resistance to tyrosine kinase inhibitors (TKIs) and advanced disease are biologically related. The hematopoietic cell cycle regulator, MS4A3, was identified as a principal component of the gene expression classifier predicting response to imatinib. Low MS4A3 correlated not only with primary TKI resistance, but also with shorter overall survival in CP-CML (n=35). Consistently, microarray (n=19 CP-CML; n=16 BP-CML), qRT-PCR (n=22 CP-CML; n=17 BP-CML), and immunoblot (n=3 CP-CML; n=3 BP-CML) analyses demonstrated that MS4A3 mRNA and protein levels are reduced in CD34+ progenitor cells from BP-CML versus CP-CML patients, with no difference between CP-CML and normal CD34+progenitors (n=3) (Eiring et al. ASH 2015 #14). These data suggest that MS4A3 may play a role in both primary TKI resistance and blastic transformation of CML. Results: To assess the functional role of MS4A3 in CML and TKI response, we used ectopic MS4A3 expression and shRNA-mediated MS4A3 knockdown in CD34+ cells from BP-CML and CP-CML patients, respectively. Ectopic expression of MS4A3 in BP-CML CD34+ progenitors (n=5) markedly reduced colony formation in the presence and absence of imatinib, consistent with a tumor suppressor role for MS4A3 in CML. While re-expression of MS4A3 alone did not increase apoptosis compared to empty vector-expressing controls, imatinib-induced apoptosis in BP-CML CD34+ cells was increased by 62%, with no effect on normal CD34+ cord blood cells (n=2). Conversely, shRNA-mediated MS4A3 knockdown (shMS4A3) in CP-CML CD34+ cells (n=7) reduced the effects of imatinib in colony formation and apoptosis assays, with no effect on normal CD34+ progenitors (n=4). In contrast to a previous report (Donato JL, et al. J Clin Invest 2002), we detected no change in cell cycle status of CML or normal CD34+ cells upon MS4A3 ectopic expression or knockdown (n=3). Altogether, these data suggest that MS4A3 positively regulates patient survival and imatinib response in CML progenitor cells. To evaluate MS4A3 in the leukemic stem cell compartment, we performed qRT-PCR on primary CP-CML cells (n=5) and observed that MS4A3 mRNA levels are 22-fold higher in committed CD34+38+ progenitors compared to more primitive CD34+38- stem cells, suggesting a role for MS4A3 in differentiation. Consistently, qRT-PCR, immunoblot, and flow cytometry demonstrated that MS4A3 mRNA and protein were upregulated in CP-CML CD34+ cells upon G-CSF treatment (n=3). Flow cytometry also revealed that shMS4A3 in CP-CML CD34+ cells resulted in a reduction of CD11b+ cells by ~45% in the presence of G-CSF (n=3). To assess the function of MS4A3 in CML stem cells, we performed long-term culture-initiating cell (LTC-IC) assays and xenografts into NSG mice upon MS4A3 knockdown in CP-CML (n=3). shMS4A3 increased Ph+ LTC-IC colony formation in the absence, and even more so in the presence, of imatinib, with no effects on Ph- LTC-ICs. Consistent with these data, shMS4A3 enhanced engraftment of CD34+CD45+GFP+ cells into the bone marrow of NSG recipient mice. Preliminary data in primary TKI-resistant and BP-CML CD34+ cells suggests regulation of this gene by promoter hypermethylation. Conclusions: Altogether, these data suggest that MS4A3 plays a key role in imatinib response of 1) patients with primary TKI resistance, 2) patients with BP-CML, and 3) the CML stem cell compartment. Since the effects of MS4A3 in CML do not involve changes to the cell cycle, experiments are underway to identify the mechanism by which MS4A3 improves imatinib response and survival in CML. Disclosures Druker: Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy. Deininger:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; 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; 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.

Background: Mutations in the BCR-ABL1 kinase domain are a well-documented mechanism of resistance to tyrosine kinase inhibitors (TKIs), but less is known about primary resistance independent of BCR-ABL1 kinase activity. We reported a gene expression classifier of TKI-naïve CD34+ cells from chronic phase chronic myeloid leukemia (CP-CML) patients that predicts cytogenetic response to imatinib (McWeeney et al. Blood 2010). The expression signature associated with primary cytogenetic failure showed overlap with previously reported signatures of blast phase CML (BP-CML), suggesting that primary TKI resistance and advanced disease are biologically similar. Results: To identify critical genes involved in primary TKI resistance, we performed principal component analysis on the expression signature and identified the hematopoietic cell cycle regulator, MS4A3, as a key factor within this classifier. Importantly, low MS4A3 expression not only correlated with primary TKI resistance, but also with shorter overall survival (p92%) in CD34+ cells from BP-CML patients (n=17; p MS4A3 expression is also low in BP-CML cell lines, including K562, KYO-1, BV-173, KCL-22, and KU-812, with the notable exception of LAMA-84 cells. Thus, to understand the functional role of MS4A3 for TKI resistance, we introduced a doxycycline-inducible shRNA targeting MS4A3 (shMS4A3) into LAMA-84 cells. qRT-PCR confirmed 50-90% MS4A3 knockdown in the presence of doxycycline (0.1 µg/mL). Consistent with its role as a tumor suppressor, MTS assays revealed that MS4A3 knockdown increased the imatinib IC50 (n=3; p2-fold upregulation of MS4A3. As expected, ectopic MS4A3 reduced colony formation by 55% in AP-CML (n=2; p Conclusion: Our results suggest that MS4A3 is a tumor suppressor protein in CML that governs TKI responsiveness and is regulated in a BCR-ABL1 kinase-independent manner. MS4A3 loss confers TKI resistance to CP-CML patients destined to exhibit primary cytogenetic failure, and in BP-CML patients with refractory resistance. MS4A3 may also contribute to the innate resistance of primitive CML stem cells. Studies to identify the mechanism of MS4A3 downregulation in TKI resistance and how its loss biochemically impairs TKI response is currently underway and will be reported. Disclosures Agarwal: CTI BioPharma: Research Funding. Deininger:BMS: Other: Consulting & Advisory Role, Research Funding; Novartis: Other: Consulting or Advisory Role, Research Funding; Celgene: Research Funding; Genzyme: Research Funding; Gilead: Research Funding; ARIAD Pharmaceutical Inc.: Other: Consulting or Advisory Role; Incyte: Other: Consulting or Advisory Role; Pfizer: Other: Consulting or Advisory Role.