ALBERT

Description: Umbilical CB is an established source of HSC for allogeneic transplantation in patients lacking HLA-matched donors. The major limitation of CB transplantation is the relatively low number HSCs within a single CB unit (CBU) resulting in delayed engraftment, infections and ultimately increased mortality. Because of this, double CBU transplantation has been used to reach the required cell doses but this approach has not led to an improved overall outcome and often results in an increased rate of GVHD. These findings have prompted the development of ex vivo expansion strategies to increase the number of HSCs, so that a single CBU can be transplanted. Most of these techniques, however, result in enrichment of short term marrow repopulating cells (ST-RC) at the expense of long term (LT)-RC which may impact durable long term engraftment. In addition, they require 2-3 weeks of culture which complicates the timing of transplant and increases the risk of contamination. Our laboratory has developed a novel approach to expand the numbers of functional HSCs, by transiently influencing the epigenetic determinants of HSCs self-renewal. A 7-day treatment of CB CD34+ cells with valproic acid (VPA) results in a dramatic increase in the number of HSCs capable of durable hematopoietic engraftment in animal model recipients (Chaurasia et al. JCI, 2014). Here we report the pre-clinical development of a VPA-expanded HSC product for utilization in the treatment of patients with hematological malignancies. In the place of using freshly collected CBU as starting sources of CD34+ cells, we validated, optimized and scaled-up the expansion procedure utilizing cryopreserved CBU procured from FDA-licensed Cord Blood Banks and clinically relevant GMP reagents and materials. CBUs from 5 different donors were subjected to thawing followed by positive CD34+ cell selection using a Miltenyi CliniMACS Prodigy®. The total number of nucleated cells (TNC), CD34+ cells, viability, clonogenic potential (i.e. CFU number) and the frequency of various HSC sub-classes were determined post-thawing after which each CBU was subjected to CD34+ cell selection. CD34+ cells counts varied between 1.6 and 13.6x106 (mean of 4.5x106/CBU) and had a purity ranging from 69.2-82.8%. CD34+ cells were treated with cytokines for 16-18h, followed by addition of VPA and ex vivo expansion for 7 days. The generated cell product was characterized phenotypically and functionally and the results were compared to the unmanipulated CBU (uCBU) (Table 1). First, the expanded grafts had greater than 90% viability (range 91.4 to 97%) as compared to 68.2% in the uCBUs after thawing. The average number of CD34+ cells generated was 494.8x106 CD34+ cells (i.e. 126-fold greater than uCBU) which is the equivalent of 61.8x105/Kg/ body weight from a single CBU for an 80 kg individual. The fraction of CD34+ cells, which represented over 60% of the expanded graft, was further assessed for the presence ST-RC, intermediate-term (IT)-RC and LT-RC defined phenotypically as CD34+/CD45RA-/CD90-/CD49f-, CD34+/CD45RA-/CD90+/CD49f-, and CD34+/CD45RA-/CD90+/CD49f+, respectively. The average numbers of each of these HSC sub-classes per expanded CBU were 64, 217 and 265 fold higher than their respective numbers found in the uCBUs. Notably, the expanded grafts contained the equivalent of 22.38x105 IT-RC/kg and 16.98x105 LT-RC/kg where as uCBUs contained only 0.1x105 IT-RC/kg and 0.06x105 LT-RC/kg. Considering the ability of these HSC sub-classes to contribute to intermediate and long term hematopoietic engraftment, their presence in such high number gives the VPA-expanded grafts improved potential to lead to durable hematopoietic and immune reconstitution after transplantation. In addition, the expanded graft has a phenotype which would also be anticipated to lead to rapid hematopoietic recovery since lineage committed precursors (i.e. CD33+, CD15+, CD235a+ and CD41+ cells) represented 35-45% of its composition. Finally, as compared to uCBUs, the expanded HSC product contained 20 times more assayable CFUs consisting predominately of CFU-GEMM which are capable of contributing to multilineage engraftment. In summary, we report the generation of an ex vivo expanded CB HSCs product highly enriched in primitive HSCs sub-classes and which is currently being developed for a Phase I clinical trial for allogeneic CB transplantation in patients with hematological malignancies. Disclosures Bhardwaj: Parker Institute of Cancer Immunotherapy: Membership on an entity's Board of Directors or advisory committees; Checkpoint Sciences: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 3453 Introduction Stathmin is a 17KDa cytosolic protein that plays an important role in the regulation of microtubule dynamics, mitotic spindle formation, cell cycle progression and cell differentiation. Stathmin knockout (KO) mice were initially reported to have a normal phenotype but were subsequently shown to develop an age-related neurological phenotype with axonopathy evident in both central and peripheral nervous systems. These mice were also shown to have a defect in recovery from acute ischemic renal injury. We had previously shown that stathmin plays an important role in the differentiation and proliferation of megakaryocytes (MK) and that down-regulation of stathmin is necessary for the maturation of MK and platelet production in vitro. In this study, we investigated the role of stathmin in megakaryopoiesis and hematopoiesis in vivo using the stathmin KO mouse as an experimental model. Results Stathmin KO mice had lower platelet (PLT) counts at 3 weeks of age when compared to WT mice. The WT mice had a mean PLT count of 662 ± 27 K/μL while KO mice had a mean PLT count of 543 ± 37 K/μL. This correlated with larger and fewer MK in the bone marrow of KO mice (WT: 4.2 ± 0.7 MK/40X field; KO: 3.6 ± 0.2 MK/40X field). Furthermore, in the spleen, there was a 10 fold decrease in the number of MK in KO mice compared to WT mice (6.6 ± 0.6 vs 0.7 ± 0.1 MK/40X field). By 8 weeks, PLT counts and MK size and numbers in the bone marrow and spleen were similar in WT and KO mice. Interestingly, by 16 weeks, the mean PLT of KO mice became significantly higher than that of WT and by 40 weeks, the mean PLT count of KO mice was 1379 ± 100K/μL compared to 1045 ± 120K/μL in WT mice (P

Description: The final stages of of megakaryocyte (MK) maturation involve a series of steps, including polyploidization and proplatelet formation. Although these processes are highly dependent on dynamic changes in the microtubule (MT) cytoskeleton, the mechanisms responsible for regulation of MTs in MKs remain poorly defined. Stathmin is a highly conserved MT-regulatory protein that has been suggested to play a role in MK differentiation of human leukemic cell lines. However, previous studies defining this relationship have reached contradictory conclusions. In this study, we addressed this controversy and investigated the role of stathmin in primary human MKs. To explore the importance of stathmin down-regulation during megakaryocytopoiesis, we used a lentiviral-mediated gene delivery system to prevent physiologic down-regulation of stathmin in primary MKs. We demonstrated that sustained expression of constitutively active stathmin delayed cytoplasmic maturation (ie, glycoprotein GPIb and platelet factor 4 expression) and reduced the ability of MKs to achieve high levels of ploidy. Moreover, platelet production was impaired in MKs in which down-regulation of stathmin expression was prevented. These studies indicate that suppression of stathmin is biologically important for MK maturation and platelet production and support the importance of MT regulation during the final stages of thrombopoiesis.

Description: 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.

Description: Abstract 548 Maturation of megakaryocytes (MK) consists of unique processes like polyploidization and proplatelet formation that are critical for efficient platelet production. Both processes are highly dependent on dynamic changes in the microtubule (MT) cytoskeleton. MT form a complex, spherical mitotic spindle that mediates polyploidization, represent the structural scaffold for proplatelet extension and enable the transport of cytoplasmic organelles into nascent platelets. While the structural organization of the MT cytoskeleton in MK is very well described at the morphological level, the mechanisms responsible for the regulation of MT dynamics during these processes are largely unknown. Stathmin is a highly conserved MT-regulatory protein that modulates MT dynamics by promoting MT depolymerization. Previous studies suggested a potential role for stathmin in MK polyploidization. However, these studies that were performed in chemically-induced transformed cell lines reached contradictory conclusions about the role of stathmin in megakaryopoiesis. We addressed this controversy by utilizing a more physiologically relevant experimental model of megakaryopoiesis in which primary human MK are grown in tissue culture. First, we optimized a MK liquid culture system that allows for efficient generation of mature MK capable of producing platelets in culture. Using this system, we demonstrated that the high levels of stathmin mRNA in proliferating MK progenitors markedly decline with maturation. We used an FIV-based lentiviral expression system to prevent this physiological downregulation of stathmin expression and investigated the effects of sustained expression on MK maturation. The sustained expression of a constitutively active form of stathmin in MK resulted in approximately 2-fold reduction in glycoprotein GPIb expression, 3.5-fold reduction in platelet factor 4 expression and loss of up-regulation of GATA-1 expression which is normally seen during maturation. This demonstrates that the sustained activity of stathmin prevents the up-regulation of three different markers of MK maturation, supporting the hypothesis that physiological downregulation of stathmin is required for proper MK differentiation. We also investigated the effects of sustained stathmin expression on polyploidization by fluorescence in situ hybridization. These studies show that stathmin inhibited the ability of MK to achieve high levels of polyploidy. Finally, we evaluated the effects of sustained stathmin expression on the ability of MK to produce platelets in vitro. We found that the number of platelets derived by MK expressing sustained levels of stathmin was reduced by approximately 50% compared to those produced by control MK. This demonstrates that sustained stathmin expression has a negative effect on platelet production, supporting the importance of MT polymerization in this process. This is the first demonstration that alterations of expression of a MT-regulatory protein can interfere with platelet production by primary MK. In summary, these studies support the importance of MT regulation during MK maturation and suggest that the physiologic downregulation of stathmin expression is biologically important in the processes of MK maturation and platelet production. Very high levels of stathmin expression are frequent in hematological malignancies such as leukemias and myelodysplastic syndrome. Inefficient platelet production is one of the most important features of these malignancies where bleeding from low platelet counts is one of the most common causes of death. Our studies open new avenues of investigation into the mechanisms responsible for MK maturation that may prove relevant for pathological conditions characterized by aberrant stathmin expression and/or function. Disclosures: No relevant conflicts of interest to declare.

Description: Imetelstat is a telomerase inhibitor which has been shown to have therapeutic activity in patients with myelofibrosis (MF) (Tefferi A. et al., 2013 ASH Annual Meeting, Abstract 662) and essential thrombocythemia (ET) (Baerlocher G. et al., 2012 ASH Annual Meeting, Abstract 179). In clinical trials involving patients with a variety of other malignancies, the primary dose-limiting toxicity of GRN163L has been thrombocytopenia. We utilized GRN163L in order to explore the possible role of telomerase on human megakaryocytes (MK) and the mechanisms underlying the drug’s inhibitory effects on platelet production. MK were generated from normal primary CD34+ cells using an ex vivo culture system previously described by our laboratory (Iancu-Rubin C. et al, Blood 2011, 117:17; 4580-4589). We first showed that both telomerase activity and the expression of its catalytic unit hTERT were elevated in proliferating normal CD34+ cells, declined transiently during the initial stages of MK differentiation but were then partially restored during the final stages of terminal maturation. Treatment of normal CD34+ cells with GRN163L did not affect the numbers of CFU-MK assayed. Furthermore, exposure to GRN163L during the first 7 days in a liquid culture system did not interfere with the ability of CD34+ cells to commit to MK. When the same cultures were allowed to mature for 7 additional days, the proportion of CD34+/CD41+ MK precursors in drug-treated cultures was twice that observed in control and the drug-treated cultures contained 70% fewer mature CD41+/CD42b+MK than control cultures. The inhibitory effects of GRN163L on MK maturation were supported by observations showing that the cultures treated with the drug contained 60% fewer polyploid MK than control cultures. These results suggest that that GRN163L-mediated inhibition of telomerase does not affect normal MK progenitors but blocks the maturation of MK precursor cells. Previous studies have shown that GRN163L inhibits neoplastic CFU-MK colony formation by CD34+ cells from patients with ET (Brunold C. et al., 2011 ASH Annual Meeting, Abstract 3843). We, therefore, examined the effects of GRN163L on malignant MK from patients with MF and ET. Unlike normal CD34+ cells, treatment of MPN PB-MNCs with GRN163L decreased the numbers of assayable CFU-MK from 6 out of 11 patients. CFU-MK colony formation by PB-MNCs from these 6 patients was reduced by 33% (ranging from 13% to 50% reduction in CFU-MK formation) as compared to PB-MNCs treated with an inactive form of the drug (p value= 0.00473). Of note, in two out of 5 patients in which the total number of CFU-MK was not affected by GRN163L treatment, the drug decreased the size of the CFU-MK colonies formed. The ability of MPN PB-MNCs to differentiate into MK was next assessed. Although the total number of cells in PB-MNC liquid cultures exposed to GRN163L was decreased as compared to those treated with the inactive drug (n=6; p value=0.03204), the proportion of CD34+/CD41+MK precursors generated was not significantly affected. We then evaluated the effects of GRN163L on the genotype of MPN MK generated in the presence and absence of GRN163L by assessing the JAK2V617F allele burden. Treatment with GRN163L but not the inactive form of the drug reduced the JAK2V617F allele burden in MK derived from two patients: in one patient, JAK2V617F allele burden in MK generated in the presence of the inactive drug was 91.86% while MK generated in the presence of GRN163L was 19.75%; MK generated from a second patient had a JAK2V617F allele burden of 10.84% in the presence of the inactive drug which was reduced to 2.14% in the presence of GRN163L. We conclude that GRN163L-mediated inhibition of telomerase affects normal megakaryopoiesis by blocking the terminal maturation of CD34+/CD41+ MK precursors, providing a possible explanation for GRN163L’s propensity to induce thrombocytopenia in patients with normal bone marrow. By contrast, GRN163L treatment inhibited the ability of MPN CD34+ cells but not normal CD34+ cells to form CFU-MK colonies and drug treatment reduced the numbers of malignant MK generated. We propose that the amelioration of fibrosis observed in a clinical trial of MF patients treated with GRN163L might be due to, at least in part, two potential modes of action: 1) inhibiting malignant MK progenitors cells and 2) preventing terminal MK maturation thus depleting the pool of mature MKs which are the major source of fibrogenic cytokines in MF. Disclosures Iancu-Rubin: Geron Corporation: Research Funding. Parker:Geron Corporation: Employment. Eng:Geron Corporation: Employment. Hoffman:Geron Corporation: Consultancy.

Description: Abstract 1082 Megakaryocyte (MK) development is characterized by polyploidization, cytoplasmic maturation and proplatelet formation, which culminates in the release of platelets into the circulation. The tumor suppressor p53 plays a critical role in the regulation of both cell cycle and apoptosis; its function is tightly controlled by the murine double minute (MDM2) protein which facilitates p53 degradation and inhibits p53 transcriptional activity. MK ploidy results from a disruption of normal cell cycle progression termed endomitosis while platelet release is believed to depend on apoptotic processes. The role of p53-MDM2 in MK in these two processes has not been clearly defined. A small molecule RG7112, which disrupts MDM2-p53 interaction, has shown promising anti-tumor effects in phase I clinical trials. This beneficial outcome has, however, been associated with the development of thrombocytopenia. We, therefore, used RG7112 as pharmacological probe to examine the effects of disruption of the MDM2-p53 regulatory loop on MK. We determined the effects of RG7112 on primary human MK by utilizing an in vitro system in which MK were generated from BM-derived CD34+ cells. We first demonstrated that both p53 and MDM2 transcripts are up-regulated as MK differentiation progresses. The ability of CD34+ cells to proliferate in the absence or presence of various concentrations of RG7112 was then evaluated both in liquid cultures and in CFU-MK colony assays. CD34+ cells exposed to 10 μM RG7112 for 7 days generated 70% fewer viable cells as compared to control cells exposed to the inactive form of the drug (p value = 0.0038). Furthermore, CD34+ cells treated with RG7112 formed up to 40% less CFU-MK colonies as compared to untreated cells. An assessment of apoptosis of MK precursors generated in the presence of RG7112 revealed that 69.5+2.1% were Annexin V positive as compared to 31.5+3.5% present in control cultures. These findings are consistent with the previously reported role of RG7112 in inducing p53 activation and apoptosis. Interestingly, phenotypical characterization of the viable cells generated under identical culture conditions, showed that RG7112 treatment did not interfere with the ability of CD34+ cells to acquire markers of MK differentiation during the first 7 days of culture since similar degrees of CD41 and CD42 expression were observed in the absence and in the presence of the drug. Likewise, exposure of MK precursors to the drug for 7 additional days (i.e. later stages of maturation) did not influence CD41 and CD42 expression. By contrast, cells differentiated in the presence of 5 μM RG7112 generated 50% fewer polyploid MK with greater than 4N DNA content as compared to those treated with the inactive form of the drug. Moreover, the negative effects on ploidy were associated with p53 activation, as assessed by the increased levels of p21 protein, a direct target of p53 which is known to limit polyploidization of primary MK. Finally, platelets generated in vitro were analyzed phenotypically and quantitated by dual labeling with anti-CD41 antibodies and thiazole orange (TO). The number of CD41+/TO+platelets derived from MK generated in the presence of RG7112 was reduced by 22% as compared to control. Based on these findings, we conclude that RG7112 impacts megakaryopoiesis by two potential mechanisms: 1) Impairing the ability of CD34+ cells to generate MK precursors due to increased apoptosis; 2) Limiting polyploidization during the late stages of development due to phamacological activation of p53. A combination of these two effects may provide an explanation for thrombocytopenia observed in patients receiving this drug and suggests that p53 plays an important role in normal human thrombocytopoiesis. Disclosures: Iancu-Rubin: Roche: Research Funding. Hoffman:Roche: Research Funding.

Description: Abstract 2610 Megakaryocytopoiesis consists of a succession of events in which MK progenitors initially proliferate and acquire lineage-specific markers, followed by polyploidization and cytoplasmic maturation. MK maturation culminates in the formation of cytoplasmic extensions (i.e. proplatelets) that leads to platelet shedding into the circulation. Panobinostat (LBH589) is a histone deacetylase inhibitor that has antiproliferative and cytotoxic effects on several types of cancer cells including blood cells from patients with hematological malignancies. One of the major adverse events associated with LBH589 treatment is thrombocytopenia. In this study, we hypothesize that the effects of LBH589 on thrombopoiesis might occur by targeting acetylation of histone and/or non-histone proteins resulting in defective platelet production. To test this hypothesis we investigated the effects of LBH589 on megakaryocytopoiesis in MK cell lines (i.e. HEL JAK2V617F positive cells) and in primary human MK. First, we tested the effects of LBH589 on the ability of human CD34+ cells to generate MK colony forming units (CFU-MKs). Neither CFU-MK or CFU-MIX derived colony formation was reduced in the presence of LBH589. To evaluate the effects of LBH589 on parameters of MK maturation, MK were generated in vitro from peripheral blood-derived CD34+ cells by employing an expansion culture system containing SCF and TPO for 6 days followed by 8 additional days incubation in the presence of TPO. These studies were pursued in the presence or absence of LBH589. Treatment with LBH589 did not significantly influence the number of CD61+ MK (i.e. control = 55.8%; 2.5nM LBH589 = 45.2%, p value=0.109; 5nM LBH589=38.5%, p value=0.095, of viable 7-AAD−/CD61+ cells) or the degree of polyploidization (i.e. control = 17.4%; 2.5nM LBH589 = 14.4%86.7, p value=0.157; 5nM LBH 589=12.8%, p value=0.116, cells with 〉4N DNA content). Culture-derived platelets were analyzed phenotypically and quantitated by means of dual labeling with anti-CD41 antibodies and with thiazole orange (TO) in order to identify new reticulated platelets. The percentage of CD41+/TO+ platelets derived from MK generated in the presence of LBH589 was significantly reduced (i.e. 2.5nM LBH589=11%, p value 0.046 and 5nM LBH589=9%, p value=0.011, CD41+/TO+ cells) as compared with MK generated in the absence of LBH589 (18.5% CD41+/TO+ cells). These findings were consistent with the observation of significant numbers of proplatelet-bearing MKs in control cultures but not in LBH 589-treated cultures. Collectively, these data suggest that LBH589 impairs platelet production while having a minimal effect on MK commitment, cytoplasmic maturation or polyploidization. To better understand the mechanisms responsible for such effects on thrombopoiesis, RNA extracted from control MK and from MK treated in vitro with LBH589 was analyzed by real time quantitative PCR to evaluate GATA-1 and NF-E2 expression. GATA-1 and NF-E2 mRNA levels were unchanged after treatment with LBH589. We found, however, that LBH589 induced a 4.8 to 7.5-fold increase in histone H3 acetylation. These data suggest that the negative impact of LBH589 on MK maturation was not mediated by its effects on chromatin but rather was possibly due to its effects on acetylation of nonhistone proteins. We demonstrated that LBH589 treatment increased acetylation of tubulin, a non-histone cytoplasmic protein that is a component of the microtubule (MT) cytoskeleton. The later stages of MK maturation are highly dependent on MT which represent the structural scaffold for proplatelet extension and enables the transport of cytoplasmic organelles into nascent platelets. The changes in the acetylation status of tubulin are critical for proper MT function and are mediated by HDAC6 which we found by Western blot analysis to be inhibited by LBH589 treatment. Based on these findings we suggest that LBH589-induced changes in tubulin acetylation result in aberrant MT function which in turn, leads to defective proplatelet and platelet formation. These nonhistone protein modifications might serve as a drug target for the development of novel agents (LBH589) to treat patients with extreme thrombocytosis due to underlying myeloproliferative neoplasms. Disclosures: Iancu-Rubin: Novartis: Research Funding. Hoffman:Novartis: Research Funding.

Description: Myeloproliferative neoplasms (MPN) are characterized by the excessive production of one or more myeloid lineages and a propensity to progress to acute leukemia. In 2013, mutations in the CALR gene, encoding calreticulin, were identified in patients with MPN, mutually exclusive to the previously identified JAK2 and MPL (TPO-R) mutations. CALR mutations are frameshift mutations - typically a 52-bp deletion (type 1) or a 5-bp insertion (type 2) - that result in a novel C-terminus. The discovery of mutations in a ubiquitously expressed multifunctional protein like calreticulin was unanticipated. Subsequent studies found that CALR mutations lead to activation of JAK/STAT, mediated through aberrant interactions between mutant CALR and MPL, thus presenting an excellent opportunity for targeted therapy. However, the mechanism of MPL activation remains largely unexplained with prior studies using cell lines with exogenous expression of CALR and MPL following transfection. To create a more physiological cellular model to study the effects of CALR mutations, we established multiple iPSC lines from two patients with CALR-mutant MPN - one type 1-like (del34) and one type 2 (ins5) -, as well as from one patient with JAK2V617F MPN. All iPSC lines were confirmed to harbour the CALR or JAK2V617F mutation found in the corresponding patient, to express mutant calreticulin, as detected by flow cytometry using an antibody which specifically recognizes the novel calreticulin C-terminus, and to be karyotypically normal. Genetically matched iPSC lines with WT JAK2 could also be generated from the JAK2V617F (but not the CALR-mutant) patient cells in the same reprogramming round. CRISPR gene editing was used to generate isogenic CALR-corrected lines from both CALR-mutant patients. Furthermore, in order to facilitate biochemical studies, we used CRISPR to introduce a V5 epitope tag in one allele of the endogenous mutant or WT CALR gene, in mutant and isogenic corrected iPSC lines, respectively. We optimized an in vitro differentiation protocol for efficient derivation of megakaryocyte (MK) progenitors from iPSCs and found disease-relevant phenotypes, mainly TPO-independent MK colony formation in semi-solid media, which is the phenotypic hallmark of ex vivo primary MPN cells. In the absence of TPO, JAK2 V617F, CALR-mutant type 1-like and CALR-mutant type 2 iPSCs generated 52.1%, 58.7±22.2% and 59.8±3.6%, respectively, of the number of MK colonies generated in the presence of TPO, as opposed to 10%, 8.8±1.8% and 0.5±0.9%, respectively, for the matched WT JAK2, the corrected CALR-mutant type 1-like and the corrected CALR-mutant type 2 iPSCs. Isolated CALR mutant iPSC-derived CD41a+ MK progenitors had increased phosphorylation of STAT5 following cytokine starvation as compared to isogenic corrected and non-isogenic normal cells. CALR-mutant cells expressed equal transcript levels of the WT and mutant CALR alleles. However, mutant CALR protein levels were severely reduced, at levels 1~12% of those of the WT protein. This is consistent with previous studies documenting instability of mutant calreticulin. Transcriptomics (RNA-seq) and proteomics analyses of CD41a+-sorted MK progenitors derived from CALR mutant and isogenic corrected iPSCs are ongoing. These iPSC models offer the opportunity to study the effects of CALR mutations in a cellular context with both MPL and CALR (WT or mutant) expressed from their endogenous loci. They thus provide a powerful platform to investigate the disease mechanisms underlying CALR-mutant MPNs and to perform small molecule and genetic (CRISPR) screens to identify new therapeutic targets. Disclosures Iancu-Rubin: Merck: Research Funding; Incyte: Research Funding; Summer Road, LLC: Research Funding; Formation Biologics: Research Funding. Hoffman:Incyte: Research Funding; Merus: Research Funding; Formation Biologics: Research Funding; Janssen: Research Funding; Summer Road: Research Funding.