Migliaccio, Anna Ritá

Abstract Background/rationale Primary myelofibrosis (PMF) and post-polycythemia vera/essential thrombocythemia (Post-PV/ET MF) (collectively termed MF) are chronic hematopoietic malignancies with progressive bone marrow collagen fibrosis, peripheral cytopenias, extramedullary hematopoiesis, splenomegaly, debilitating constitutional symptoms and heightened risk of transformation to acute leukemia. The only currently approved therapies for MF are JAK inhibitors (JAKi) which can provide significant improvements in splenomegaly and systemic symptoms but do not fundamentally change the pathogenesis of the disease. As a result, additional non-JAKi therapies for patients with MF after JAKi failure represent an important translational focus. Reparixin is a novel, potent and specific inhibitor of the chemokine CXCL8 (IL)-8. IL-8 is a member of a class of cytokines involved in leukocyte recruitment and activation in tissues. There are two known types of CXCL8 receptors – CXCR1 and CXCR2, which are expressed in neutrophils (Baggiolini M, Adv Immunol 1994). IL-8/CXCR1/2 signaling has been shown to be a key mediator of inflammatory signaling in MF patients. Hematopoietic stem/progenitor cells from patients with MF are enriched for a CXCL8/CXCR2 gene signature and display enhanced proliferation and fitness in response to an exogenous CXCL8 ligand in vitro. Genetic deletion or a small molecule inhibitor of CXCR2 in MF mouse adoptive transfer models ameliorates fibrosis, extends overall survival and improves hematologic parameters (Dunbar A, Blood 2023). This pre-clinical data validates targeting this pathway in clinical investigations. Study design This is an open-label, phase II study assessing the efficacy, safety, and tolerability of reparixin in patients with Dynamic International Prognostic Scoring System (DIPSS) intermediate-2, or high-risk PMF, post ET/PV MF either after prior treatment with, or those who are ineligible for treatment with a JAKi. Patients will receive oral reparixin 1200 mg three times daily on a four-week cycle for a core study period of 24 weeks. Patients who achieve at least clinical improvement (CI) or stable disease (SD) with one grade improvement in bone marrow fibrosis by International Working Group-Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) criteria at the Cycle 7 response assessment may continue receiving reparixin until loss of response, disease progression, unacceptable toxicity, patients/physician withdrawal, or termination of study by sponsor. The study monitors both efficacy and toxicity using a Bayesian Optimal Phase 2 (BOP2) design. If response is observed in at least one patient out of 10 and safety is confirmed at the interim analysis, the trial will proceed to full enrollment of 26 patients. If more than three responses are observed in the total cohort, then a signal of activity is confirmed. Primary Objective: To estimate the efficacy of reparixin treatment in DIPSS intermediate-2 or high-risk subjects with PMF, post PV-MF, or post ET-MF. Secondary Objectives: To assess the efficacy of reparixin as determined by response assessment using IWG-MRT/ELN criteria at the end of Cycle 6 and Cycle 12. To assess the change in bone marrow fibrosis grade at the end of Cycle 6 and Cycle 12 with reparixin. To assess the safety of reparixin as measured by the adverse event profile of Common Terminology Criteria for Adverse Events v5.0. To assess spleen volume reduction by imaging after Cycle 6 and Cycle 12 as compared to baseline spleen volume. Key Exploratory Objectives: To evaluate changes in quality of life and patient-reported outcomes To conduct correlative studies to measure biomarkers of reparixin activity, such as Vascular Endothelial Growth Factor (VEGF) levels, bone marrow microvessel density, megakaryocyte number and peripheral blood CD34+ cell number, and patients with and without IL-8 secretion. Key inclusion criteria: Have a pathologically confirmed diagnosis of PMF, post-ET-MF, or post-PV-MF as per the WHO diagnostic criteria with intermediate-2 or higher risk disease by DIPSS Be refractory/resistant to or intolerant of/inappropriate for JAKi therapy Have adequate liver and kidney function Platelet count ≥ 25 x 109/L Bone marrow and peripheral blood blast count < 10% ANC ≥ 1000 mm3. MPN Research Consortium (MPN-RC) 120 is currently enrolling patients through the MPN-RC clinical sites throughout the United States. Clinical Trial Identifier NCT05835466

Abstract Purpose: Myelofibrosis (MF) is a clonal myeloproliferative neoplasm characterized by systemic symptoms, cytopenias, organomegaly, and bone marrow fibrosis. JAK2 inhibitors afford symptom and spleen burden reduction but do not alter the disease course and frequently lead to thrombocytopenia. TGFβ, a pleiotropic cytokine elaborated by the MF clone, negatively regulates normal hematopoiesis, downregulates antitumor immunity, and promotes bone marrow fibrosis. Our group previously showed that AVID200, a potent and selective TGFβ 1/3 trap, reduced TGFβ1-induced proliferation of human mesenchymal stromal cells, phosphorylation of SMAD2, and collagen expression. Moreover, treatment of MF mononuclear cells with AVID200 led to increased numbers of progenitor cells (PC) with wild-type JAK2 rather than JAK2V617F. Patients and Methods: We conducted an investigator-initiated, multicenter, phase Ib trial of AVID200 monotherapy in 21 patients with advanced MF. Results: No dose-limiting toxicity was identified at the three dose levels tested, and grade 3/4 anemia and thrombocytopenia occurred in 28.6% and 19.0% of treated patients, respectively. After six cycles of therapy, two patients attained a clinical benefit by IWG-MRT criteria. Spleen and symptom benefits were observed across treatment cycles. Unlike other MF-directed therapies, increases in platelet counts were noted in 81% of treated patients with three patients achieving normalization. Treatment with AVID200 resulted in potent suppression of plasma TGFβ1 levels and pSMAD2 in MF cells. Conclusions: AVID200 is a well-tolerated, rational, therapeutic agent for the treatment of patients with MF and should be evaluated further in patients with thrombocytopenic MF in combination with agents that target aberrant MF intracellular signaling pathways.

Abstract TGFβ plays a pivotal role in the pathobiology of myelofibrosis (MF) by not only promoting bone marrow fibrosis (BMF) but also by enhancing the dormancy of normal but not MF hematopoietic stem cells (HSCs). TGFβ has also previously been reported to inhibit normal megakaryocyte (MK) production (Bruno et al Blood 1998). TGFβ1 promotes the synthesis of collagen by normal human mesenchymal stromal cells (MSCs). Treatment of MSCs with AVID200, a potent TGFβ1/3 protein trap, significantly decreased MSC proliferation, phosphorylation of SMAD2, and collagen expression. Robust expression of pSMAD2 was observed in the absence of exogenous TGFβ in normal donor or MF-MKs, Addition of AVID200 to MKs decreased pSMAD2 without affecting total SMAD2/3 and led to increased numbers of MKs. Treatment of MF MNCs with AVID200 also led to increased numbers of progenitor cells with wild type JAK2 and a reduction of mutated colonies. A phase 1b trial of AVID200 (NCT03895112) was performed and completed in INT-2/high risk MF patients resistant/intolerant to ruxolitinib (rux); baseline platelet count of ≥ 25 x 10 9/L, and grade 2/3 BMF. Subjects received AVID200 intravenously on Day 1 of a 21 day cycle. Response was assessed by IWG/ELN criteria after 6 cycles of AVID200. Subjects attaining at least a CI or SD with a decrease in BMF by ≥1 grade, continued AVID200. We previously presented the results of the dose escalation study (Mascarenhas ASH 2020) demonstrating that AVID200 was well tolerated without dose limiting toxicities at 3 tested dose levels (Lots A and B) in dose cohorts of 180 mg (A), 550 mg (A)/70 mg (B), and 180 mg (B). Here we report updated safety and efficacy results of the phase 1b dose expansion stage at the two highest doses tested (70 mg (B) and 180 mg (B). Twenty-two subjects were enrolled (1 withdrew before receiving treatment) and 9 were treated with AVID200 in the dose escalation phase and 12 in the dose expansion phase [Table1]. Median time after rux discontinuation was 7.4 months (0.5-59.9). The most common mutations observed at baseline in this cohort included JAK2V617F (71%), TET2 (29%) ASXL1 (24%) and CALR (19%). (Fig 1) No DLTs were observed and Grade 3/4 AEs were observed in 16 (76.2%) subjects. Grade 3/4 non-hematologic AEs were observed in 8 (38.1%) subjects and included one subject in each case (epistaxis, mucositis, extraocular muscle paresis, fatigue, rash, duodenal hemorrhage, gastric hemorrhage, urinary tract infection, and syncope). Grade 3/4 hematologic AEs were anemia (6; 28.3%) and thrombocytopenia (2; 14.3%) [Table 2]. No fatal events were observed. The median number of cycles received was 5 (range 2 - 13) and 7 (33%) patients received more than 6 cycles. For dose levels 2-3 at cycle 7, a CI was attained in one subject at dose level 2 [anemia, spleen and TSS], 5 subjects had SD, 3 subjects had PD and two subjects with 10% and 15% blasts at screening developed MPN-BP while on study based on central review. Reasons for discontinuation by local PI included PD (n=8), lack of response (n=5), study completed (n=2), other (n=2), patient decision (n=1). Median % change in palpable spleen length was +10% (range -70% to +150%) and TSS change was -50% (-100% to +185.7%) The median platelet count at baseline was 114 x 10 9/L (range: 28-695) and 215 x 10 9/L (range: 66-263) after cycle 6 in 7 evaluable subjects (Fig 2A). Notably, 17 subjects had an increase in platelets from baseline during treatment and two subjects normalized their platelet counts. Maximum changes in platelets from baseline across all cycles was +63.8% [range -15.7%, +505.5%] (Fig 2B). Paired bone marrow biopsy pathology samples for 12 subjects were available for central review and showed no significant changes in BMF score or MK histo topography at end of treatment compared to baseline. All patients had elevated plasma levels of TGF β1, but not TGFβ2/β3 levels as detected by ELISA, which were dramatically reduced 21 days after the last dose of AVID200. AVID200 a TGFβ1/3 protein trap is well tolerated and clinical responses at cycle 7 of therapy in this advanced MF patient population were limited as judged by IWG/MRT response criteria. However, AVID200 therapy resulted in significant reduction in serum TGFβ levels and improvements in platelet counts indicating that TGF β1 plays a pivotal role in MF leading to thrombocytopenia which can be reversed with AVID200 therapy. We conclude that AVID200 may best be employed in combination therapy approaches in thrombocytopenic MF patients. Figure 1 Figure 1. Disclosures Mascarenhas: Constellation: Consultancy, Membership on an entity's Board of Directors or advisory committees; Promedior: 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, Research Funding; Geron: Consultancy, Research Funding; Forbius: Research Funding; Genentech/Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Galecto: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Prelude: Consultancy; Kartos: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI Biopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Geron: Consultancy; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merus: Research Funding. Palmer: PharmaEssentia: Research Funding; Sierra Oncology: Consultancy, Research Funding; Incyte: Research Funding; CTI BioPharma: Consultancy, Research Funding; Protagonist: Consultancy, Research Funding. Kuykendall: Celgene/BMS: Honoraria; Pharmaessentia: Honoraria; Novartis: Honoraria, Speakers Bureau; Protagonist: Consultancy, Research Funding; Incyte: Consultancy; Abbvie: Honoraria; Blueprint: Honoraria. Mesa: Genentech: Research Funding; Promedior: Research Funding; Samus: Research Funding; Gilead: Research Funding; CTI: Research Funding; Abbvie: Research Funding; Sierra Oncology: Consultancy, Research Funding; Celgene: Research Funding; Novartis: Consultancy; Pharma: Consultancy; CTI: Research Funding; Constellation Pharmaceuticals: Consultancy, Research Funding; AOP: Consultancy; La Jolla Pharma: Consultancy; Incyte Corporation: Consultancy, Research Funding. Rampal: Stemline: Consultancy, Research Funding; Memorial Sloan Kettering: Current Employment; BMS/Celgene: Consultancy; Abbvie: Consultancy; CTI: Consultancy; Novartis: Consultancy; Disc Medicine: Consultancy; Blueprint: Consultancy; Pharmaessentia: Consultancy; Incyte: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Constellation: Research Funding; Kartos: Consultancy; Sierra Oncology: Consultancy. Gerds: PharmaEssentia Corporation: Consultancy; Sierra Oncology: Consultancy; CTI BioPharma: Research Funding; Constellation: Consultancy; Celgene/Bristol Myers Squibb: Consultancy; AbbVie: Consultancy; Novartis: Consultancy. Yacoub: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees; ACCELERON PHARMA: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Dynavex: Current equity holder in publicly-traded company; Cara: Current equity holder in publicly-traded company; Ardelyx: Current equity holder in publicly-traded company; Seattle Genetics: Honoraria, Speakers Bureau; Incyte: Consultancy, Honoraria, Speakers Bureau; Hylapharm: Current equity holder in publicly-traded company. Talpaz: Imago: Consultancy; Constellation: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Other: Grant/research support ; Celgene: Consultancy. Komrokji: Acceleron: Consultancy; Taiho Oncology: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy; BMSCelgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PharmaEssentia: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Geron: Consultancy; Jazz: Consultancy, Speakers Bureau. Kremyanskaya: Astellas: Research Funding; Astex: Research Funding; Chimerix: Research Funding; Bristol Myers Squibb: Research Funding; Constellation: Research Funding; Protagonist Therapeutics: Consultancy, Research Funding; Incyte: Research Funding. Salama: Mayo Clinic: Current Employment, Other: Mayo Clinic had the contractual work for the central pathology review for this study and I was one of the reviewing pathologists; Constellation Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Hoffman: Kartos Therapeutics, Inc.: Research Funding; Protagonist Therapeutics, Inc.: Consultancy; Novartis: Other: Data Safety Monitoring Board, Research Funding; AbbVie Inc.: Other: Data Safety Monitoring Board, Research Funding. OffLabel Disclosure: AVID200 is a TGFb trap and is in clinical testing for fibrotic diseases. It does not have an approved indication at this time.

Idiopathic pulmonary fibrosis (IPF) is a chronic inflammatory disorder which leads to progressive fibrosis and lung failure within 2-5 years1,2. Approximately 30% of IPF patients develop Chronic Kidney Disease (CKD) with renal dysfunction and fibrosis3. The treatment options for IPF are still limited and the disease is an unmet clinical need. The only animal model for IPF is represented by mice treated with bleomycin, a compound used for chemotherapy which has lung toxicity as its major adverse effect, that develop progressive fibrosis. Recent data indicate that bleomycin triggers lung fibrosis by increasing the release of the pro-inflammatory cytokine TGF-β by the resident megakaryocytes (MK)4. Using these observations as foundation, we investigated whetherGata1lowmice, a model for myelofibrosis induced by a TGF-β/P-selectin (P-sel) circuitry established by the abnormal MK5, includes fibrosis in lungs and kidney and whether these traits are rescued by genetic ablation (P-selnullmutation) and antibody neutralization (by RB40.34) of P-sel. MK were observed in lungs from wild-type (WT, n=9),Gata1low(n=22),P-selnull(n=4) andP-selnullGata1low(n=10) littermates. In frequency, MK were significantly lower inGata1low andP-selnullGata1lowthan in WT andP-selnulllittermates (median=1.3-1.5 vs 3-3.7/0.144mm2, p<0.003) and mostly immature. In addition, lungs and kidney fromGata1low mice, but not those from the other experimental groups, contained great levels of TGF- β (Fig.1). Interstitial pneumonia was observed in lungs from WT (n=12, age=3-13 months, males=8, females=4),P-selnull(n=9, age=3-16 months, males=4, females=5),Gata1low(n=29, age=2-24 months, males=16, females=13) andGata1lowP-selnull(n=18, age=3-16 months, males=7, females=11) mice while nephritis was observed only in kidneys fromGata1low mice. Controlling for the effects of sex, there are statistically significant differences in severity of inflammation in kidney (p=0.001) but not in lungs (p=0.86) betweenGata1low and WT mice. These results indicate that lung inflammation is a baseline condition of the CD1 background used for the study while kidney inflammation is a trait exclusive toGata1low mutants independently from their gender. Great levels of fibrosis were observed in lungs and kidney fromGata1low mice but not in those from WT littermates. There are statistically significant differences in lung (p=0.01) and kidney (p<0.001) fibrosis between the two groups; these differences remained after adjusting for age or sex. By contrast, there is insufficient evidence to conclude there exists a relationship between age and fibrosis grade in the other experimental groups. Age was found to be a statistically significant effect modifier on the relationship between genotype and fibrosis in lung (p=0.024) and kidney (0.004). In addition, although there is insufficient evidence for a relationship between sex and fibrosis grade in lung (p=0.62), there is a statistically significant effect of gender in kidney fibrosis inGata1low mice (p<0.001). These results indicate that fibrosis in lung and nephritis and fibrosis in kidney are novel traits ofGata1low mice developed independently from the underlying state of inflammation and which are rescued by ablation ofP-sel. To further clarify the role of P-sel in lung and kidney fibrosis, 14Gata1lowmice (7 males, 7 females) were treated with either vehicle (8 mice) or RB40.34 (30 μg/mouse/day per 3 days/week by tail vein injection as described6). Mice were sacrificed on day 5 (males) and 12 (females) and histopathology, inflammation, fibrosis and TGF-β content in lungs and kidney analyzed. Treatment with RB40.34 restored the morphology and reduced inflammation, fibrosis and TGF-β content in the lungs and in kidney with respect to vehicle (Fig.1). In conclusion, Gata1low mice are a novel animal model for IPF with CKD. The phenotype is associated with highTGF-βcontent in lung and kidney and is rescued by targeting P-sel, suggesting that treatment with antibodies against P-sel may cure IPF in man. REF: 1Richeldi et al.Lancet389: 1941, 2017; 2Hutchinson et al.EurRespirJ46, 795, 2015; 3Ikezoeet al.Respiration94: 346, 2017; 4Zhouet al.CellDeathDis10:648, 2019: 5Spangrudeet al.StemCells34: 67, 2016; 6Emburyet al.Blood104: 3378, 2004. Disclosures Migliaccio: Novartis:Research Funding.

Preclinical Rationale: Myelofibrosis (MF) is a chronic myeloproliferative neoplasm for which there are limited therapies. TGFβ plays a pivotal role in the pathobiology of MF by not only promoting bone marrow fibrosis (BMF) and collagen deposition, but also by enhancing the dormancy of normal but not MF hematopoietic stem cells (HSCs). TGFβ has also previously been reported to inhibit normal megakaryocyte (MK) production (Bruno et al Blood 1998). TGFβ1 promotes the synthesis of collagen by normal human mesenchymal stromal cells (MSCs) and activates the TGFβ receptor I/SMAD pathway as well as non-canonical TGFβ pathways. We generated MKs from MF subject mononuclear cells (MNCs) and showed that they elaborated significantly greater levels of TGFβ1 than TGFβ2/3 TGFβ1 treatment reduced the numbers of hematopoietic colonies generated by normal but not MF MNCs. Treatment of MSCs with AVID200, a potent TGFβ1/3 protein trap, significantly decreased MSC proliferation, phosphorylation of SMAD2, and collagen expression. Robust expression of pSMAD2 was observed in the absence of exogenous TGFβ in normal donor or MF-MKs, Addition of AVID200 to -MKs decreased pSMAD2 without affecting total SMAD2/3, indicating that AVID200 blocks the effects of autocrine TGFβ produced by MKs and led to increased numbers of MKs. Moreover, treatment of primary MF MNCs with AVID200 led to increased numbers of progenitor cells with wild type JAK2 and a reduction of mutated colonies. AVID200 blocked TGFβ1-induced p57Kip2 expression and SMAD2 activation by MF MNCs allowing the normal progenitor cells to preferentially cycle, proliferate, and form hematopoietic colonies. Clinical Trial Design: Based on these findings, a phase 1 trial of AVID200 is ongoing in INT-2/high risk MF subjects resistant or intolerant to ruxolitinib; baseline platelet count of ≥ 25 x 109/L, and grade 2/3 BMF. Subjects received intravenous AVID200 (Lots A and B) in dose cohorts of 180 mg/m2 (A), 550 mg/m2 (A), 180 mg/m2 (B) on Day 1 of a 21 day cycle. Cohorts of 3 subjects with a target toxicity rate of 30% were enrolled to estimate the maximum tolerated dose (MTD). A modified toxicity probability interval design was used. Response was assessed by IWG/ELN criteria after 6 cycles of AVID200. Subjects attaining at least a CI or SD with a decrease in BMF by ≥1 grade, continued AVID200. Clinical Trial Results: 10 subjects were enrolled (1 withdrew before receiving treatment) and 9 were treated with AVID200 and were evaluable for DLT assessment [Table1]. Median time after ruxolitinib discontinuation was 3.5 months (0.5-12.2). No DLTs were observed. Grade 3/4 AEs (regardless of attribution) were observed in 6 (66.7%) subjects. Grade 3/4 non-hematologic AEs observed were epistaxis (1, 11.1%), extraocular muscle paresis (1, 11.1%), fatigue (1, 11.1%) and rash (1, 11.1%). Grade 3/4 hematologic AEs were anemia (3, 33.3%) and thrombocytopenia (2, 22.2%) [Table 2]. The median number of cycles received was 5.7 (range 0 - 12). 5 subjects received 6+ cycles and were evaluable. CI occurred in 2 subjects [anemia, spleen and TSS (n=1); TSS (n=1)] 1 of which is still being treated, 2 subjects had SD, 1 subject with 21% blasts prior to study treatment had progressive MPN-BP. 4 subjects failed to reach response evaluation after 6 cycles, 2 had PD due to increasing splenomegaly, 1 subject received an allogeneic transplant and 1 is still being treated [Cycle 2]. The median platelet count at baseline was 114 (range: 42-290) and 159 after cycle 6 [Figure 1]. Maximum changes in platelets from baseline was +64% [range -73%, 169%] in all subjects. 7 subjects had an increase in platelets from baseline during treatment. 2 subjects normalized their platelet count from thrombocytopenic levels. The effect of AVID200 on BMF is currently being examined. 2 subjects remain on treatment. Conclusions: AVID200 a TGFβ1/3 protein trap is well tolerated in advanced MF subjects. Clinical responses were observed at the 550 mg dose and the expansion efficacy cohorts at doses 2 and 3 are enrolling 12 additional subjects. Furthermore, AVID200 therapy improved thrombocytopenia in MF subjects which may be due to AVID200 inhibiting the effects of TGFβ1 on normal MKpoiesis. Updated subject safety and efficacy data along with correlative data will be presented. 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). Kuykendall:Blueprint Medicines: Research Funding; BMS: Research Funding; Incyte: Research Funding; Novartis: Research Funding. Komrokji:Jazz: Honoraria, Speakers Bureau; Abbvie: Honoraria; Agios: Speakers Bureau; BMS: Honoraria, Speakers Bureau; Geron: Honoraria; Incyte: Honoraria; Acceleron: Honoraria; Novartis: Honoraria. Gerds:Gilead Sciences: Research Funding; Imago Biosciences: Research Funding; Sierra Oncology: Research Funding; Celgene: Consultancy, Research Funding; Roche/Genentech: Research Funding; CTI Biopharma: Consultancy, Research Funding; Apexx Oncology: Consultancy; AstraZeneca/MedImmune: Consultancy; Pfizer: Research Funding; Incyte Corporation: Consultancy, Research Funding. Migliaccio:Novartis: Research Funding. O'Connor-McCourt:Forbius: Current Employment. Tremblay:Forius: Current Employment. Nadler:Forbius: Consultancy; Nadler Pharma Associates: Current Employment; Symphogen: Consultancy; Iksuda Therapeutics: Consultancy; Tessa Therapeutics: Consultancy. Mesa:Celgene: Research Funding; Genetech: Research Funding; Samus: Research Funding; Promedior: Research Funding; CTI: Research Funding; LaJolla Pharma: Consultancy; Incyte: Research Funding; Sierra Onc: Consultancy; Abbvie: Research Funding; Novartis: Consultancy. Hoffman:Forbius: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Dompe: Research Funding; Protagonist: Consultancy.

Abstract The inhibitory effects exerted by transforming growth factor-β (TGF-β) on adult erythropoiesis has been well established. Canonical SMAD-depedent TGF-β signaling inhibits erythroid differentiation at multiple levels: it induces hematopoietic stem cells into quiescence (Chabanon et at Stem Cells. 2008;26:3150), it elicits a Smad5-dependent inhibition of progenitor cell proliferation (Bruno et al, Blood 1998; 91:1971) by increasing the length of G1 through reduction of G1 cyclin and cyclin-dependent protein kinases (Jacobsen et al, Blood 1995; 86:2957; Geng and Weinberg, PNAS 1993; 90:10315) and triggers Smad4-signaling accelerating terminal erythroid maturation (Zermati et al, Exp hematol 2000; 28:885; Choi et al MCB 2005;271:23). TGF-β is expressed by almost every cell type, but is produced in the greatest amount by megakaryocytes (Assonian et al, JBC 1983;258:7155; Massague, Cell 2008;134, 215). TGF-β has been implicated in the pathogenesis of primary myelofibrosis (PMF) by Schmitt et al (Blood. 2000;96:1342). Plasma, bone marrow and spleen washes of PMF patients, as well as those from murine PMF models, contain 2 fold-greater levels of total and bioactive TGF-β than those from normal controls (Zingariello et al, Blood 2013;121:3345). In addition, megakaryocytes from PMFpatients contain greater amounts of TGF-β than those from other MPNs such as polycythemia vera (PV) and essential thrombocythemia (Ciurea et al, Blood 2007;110:986). Published data, however, also suggest that malignant MPN cells are insensitive to TGF-β. Microarray analyses of PMF bone marrow and spleen cells revealed TGF-β signaling abnormalities which predict activation of non-canonical p38/ERK-dependent rather than canonical SMAD-dependent signaling (Ciaffoni et al, BCMD 2015;54:234). In addition, by phosphoproteomic profiling of PV erythroblasts (Erys) expanded in vitro express lower levels of pSmad2 as compared to Erys from healthy controls (Hricik et al. AJH 2013;88:723). The hypothesis that malignant MPN cells are insensitive to TGF-β was tested here by evaluating the effect of SB431542 [1,3,10 and 26µM], a small molecule inhibitor of TGF-β receptor 1, on ex vivo erythropoiesis in cultures generated by peripheral blood mononuclear cells from patients with JAK2 V617+-PV (n=2) and JAK2 V617F+ or CALR pQ365f+-PMF. Identical experiments were performed with Erys generated from adult peripheral blood (AB, n=3) and cord blood (CB, n=2) of healthy controls. All cultures were stimulated with SCF, IL-3 and EPO with and without dexamethasone (Dex). In cultures of JAK2 V617F+ -PV, SB431542 increased by 2-fold the numbers of progenitor cells observed by day 6, but had no effect on that of Erys observed by day 12-17 [fold increase (FI) ~4 fold in all cases]. Moreover, neither the number of progenitor cells nor that of Erys were affected by SB431542 treatment in cultures generated from JAK2 V617F+ (n=1) and CALR pQ365fs+ -PMF (n=1) patients (Fig. 1). This lack of effects was observed in cultures with and without Dex. By contrast, as expected, in cultures of AB, SB431542 significantly increased by 2.5-fold the number of progenitor cells observed by day 6 and that of Erys observed by day 14-17 (Fig. 1). This increase was associated with greater retention of an immature erythroid phenotype (CD36+ CD235a+ cells 16% vs 3%) and an increased proliferative index (>3 Erys in metaphases per field vs 0). This was observed up to day 17 in cultures both with and without Dex. The effects of SB431542 in cultures of CB were, however, affected by the presence of Dex. In cultures without Dex, SB431542 increased by 2-fold the number of progenitor cells by day 6 but had no effect on that of Erys by day 12-17 (FI=10-15 and CD36+CD235a+ cells >60%). In the presence of Dex, SB431542 did not affect the number of progenitor cells at day 6 but reduced that of Erys by 3-fold on day 12-17. These results suggest that in the case of CB, TGF-β promotes erythroid maturation in synergy with Dex. In conclusion, SB431542 promoted proliferation and maturation of normal adult progenitor cells but had no effect on PMF progenitor cells suggesting that treatments with TGF-β receptor 1 inhibitors may reactivate normal hematopoiesis in PMF patients by providing a proliferative advantage to the resident non-diseased hematopoietic stem cells over the malignant clone. This therapeutic approach will be explored in a MPD-RC, multi-center, phase II trial in patients with PMF. Disclosures Hoffman: Geron: Consultancy, Membership on an entity's Board of Directors or advisory committees; All Cells, LLC: Consultancy, Membership on an entity's Board of Directors or advisory committees; Promedior: Research Funding. Mascarenhas:Roche: Research Funding; Incyte Corporation: Research Funding; Kalobios: Research Funding; CTI Biopharma: Research Funding; Novartis Pharmaceuticals Corporation: Research Funding; Promedior: Research Funding.

On March 25 and 26, 2015, the National Heart, Lung, and Blood Institute sponsored a meeting on the State of the Science in Transfusion Medicine on the National Institutes of Health (NIH) campus in Bethesda, Maryland, which was attended by a diverse group of 330 registrants. The meeting's goal was to identify important research questions that could be answered in the next 5 to 10 years and which would have the potential to transform the clinical practice of transfusion medicine. These questions could be addressed by basic, translational, and/or clinical research studies and were focused on four areas: the three “classical” transfusion products (i.e., red blood cells, platelets, and plasma) and blood donor issues. Before the meeting, four working groups, one for each area, prepared five major questions for discussion along with a list of five to 10 additional questions for consideration. At the meeting itself, all of these questions, and others, were discussed in keynote lectures, small‐group breakout sessions, and large‐group sessions with open discourse involving all meeting attendees. In addition to the final lists of questions, provided herein, the meeting attendees identified multiple overarching, cross‐cutting themes that addressed issues common to all four areas; the latter are also provided. It is anticipated that addressing these scientific priorities, with careful attention to the overarching themes, will inform funding priorities developed by the NIH and provide a solid research platform for transforming the future practice of transfusion medicine.