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Background: Clinical outcomes for children with FLT3-mutant AML and infants with KMT2A-rearranged (KMT2A-R) B-ALL remain dismal. These leukemias share a common feature of aberrant activation of FLT3 kinase signaling, which occurs by activating FLT3 mutations in AML and by overexpression of wild-type FLT3 in KMT2A-R ALL. Several FLT3 tyrosine kinase inhibitors (FLT3i) are approved for adults with FLT3-mutant AML, but potential efficacy against KMT2A-R ALL remains incompletely characterized and may differ from responses in AML. We previously developed and preclinically validated chimeric antigen receptor (CAR) T cells directed against FLT3 (FLT3CART), which importantly showed potent anti-leukemia activity in preclinical models of both childhood FLT3-mutant AML and infant KMT2A-R ALL (Chien CD et al. ASH 2016). In the current studies, we hypothesized that combinatorial targeting of these two high-risk leukemia subtypes with FLT3CART and the selective next-generation FLT3i gilteritinib would have superior activity and potentially mitigate therapeutic resistance now known to occur with kinase inhibitors or CAR T cell immunotherapy. Methods and Results: We first assessed in vitro sensitivity of human FLT3-mutant AML and KMT2A-R ALL cell lines to gilteritinib, a second-generation selective FLT3i with established clinical activity in FLT3-mutant AML and unknown activity in KMT2A-R ALL. As detrimental effects of kinase inhibitors (e.g., dasatinib, ruxolitinib) upon CAR T cells have been reported, we evaluated for similar effects with gilteritinib co-incubated in vitro with CD3/CD28-bead activated healthy human donor T cells. However, we observed minimal deleterious effects of gilteritinib on normal T cell viability, immunophenotype, and IL-2 and interferon-gamma (IFNg) production. We validated combinatorial effects of gilteritinib and FLT3CART-induced cytotoxicity against FLT3-mutant AML and KMT2A-R ALL cell lines in vitro without impairment of IL-2/IFNg production. We then assessed this dual therapy approach in luciferase+ FLT3-mutant AML (MOLM14) and KMT2A-R ALL (SEM) cell line murine xenograft models. As predicted, both FLT3CART and gilteritinib monotherapies transiently inhibited in vivo leukemia proliferation, although leukemia progression eventually occurred. Conversely, FLT3CART and gilteritinib combination therapy strikingly induced enhanced and sustained leukemia clearance in all assessed AML and ALL cell line xenograft models (Figure 1). Confirmatory studies in our established childhood FLT3-mutant AML and KMT2A-R ALL patient-derived xenograft (PDX) models have also demonstrated potent anti-leukemia efficacy of combined FLT3CART and gilteritinib therapy. Earlier-generation FLT3i have been reported to increase cell surface FLT3 expression on FLT3-mutant AML cells. Given the known importance of target antigen site density for CAR T cell efficacy, we reasoned that a sequential approach to dual therapy with FLT3i 'priming' followed by FLT3CART may be superior to a simultaneous treatment strategy. In vitro studies with leukemia cell lines and in vivo studies with PDX models indeed confirmed gilteritinib-induced increases in FLT3 surface antigen density in FLT3-mutant AML cells. Intriguingly, we observed contrasting effects in KMT2A-R ALL cell lines and PDX with decreased surface FLT3 expression upon gilteritinib exposure. Ongoing studies are currently validating gilteritinib priming for FLT3CART given these initial data suggesting potentially divergent sequencing approaches in FLT3-mutant AML versus KMT2A-R ALL. Conclusions: Taken together, our preclinical studies demonstrate that dual targeting with FLT3CART immunotherapy and gilteritinib is a promising therapeutic strategy in FLT3-mutant AML and, importantly, also in KMT2A-R ALL. Notably, we also report minimal negative effects of gilteritinib on FLT3CART, suggesting that FLT3i may be used to enhance CAR T cell immunotherapy without inhibiting T cell function. Phase 1 clinical trials of FLT3CART will open soon for adults and children with FLT3-mutant AML and/or KMT2A-R ALL. Figure 1 Figure 1. Disclosures Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company; ElevateBio: Research Funding. Tasian: Kura Oncology: Consultancy; Aleta Biotherapeutics: Consultancy; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding.

Introduction : Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is associated with high rates of chemoresistance and relapse. CRLF2 (cytokine receptor-like factor 2) rearrangements occur in 50% of Ph-like and 60% of Down Syndrome (DS)-associated ALL and induce constitutive JAK/STAT and other kinase signaling. Current clinical trials are studying chemotherapy with the JAK inhibitor ruxolitinib in patients with CRLF2-rearranged Ph-like ALL, but results are not yet known. While chimeric antigen receptor T-cell (CART) immunotherapies have induced remarkable remissions in children with relapsed/refractory B-ALL, approximately 50% of CD19CART-treated patients will relapse again, many with CD19 antigen loss. New therapies are needed to prevent relapse and overcome immunotherapeutic resistance. Methods : We previously developed CAR T cells targeting the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) and demonstrated potent preclinical activity in Ph-like ALL models (Qin Blood 2015), which has led to a soon-to-open phase 1 clinical trial for patients with relapsed/refractory CRLF2-overexpressing ALL. In the current preclinical studies, we hypothesized that combinatorial targeting with bispecific TSLPRxCD19CART or TSLPRxCD22CART (Ross Cancer Res 2020) or with TSLPRCART + ruxolitinib will have superior activity against CRLF2-rearranged Ph-like and DS-ALL. Results : TSLPRCART treatment of CRLF2-rearranged ALL cell line (n=1) and patient-derived xenograft (PDX) models potently inhibited leukemia proliferation in vitro and in vivo and induced long-term 'cure' of xenograft mice. However, co-administration of TSLPRCART + ruxolitinib markedly diminished in vivo T cell numbers, blunted cytokine production, and facilitated leukemia relapse, which could be abrogated by delaying ruxolitinib. Importantly, ruxolitinib co-treatment prevented severe TSLPRCART-induced cytokine release syndrome (CRS) and animal death. Interestingly, ruxolitinib withdrawal led to return of T-cell functionality with re-detection of TSLPRCART in peripheral blood, induction of IFN-γ production, and leukemia clearance upon CRLF2+ ALL rechallenge (Figure 1). Conclusions: In these preclinical studies, we report potent activity of TSLPRCART in cell line (n=1) and PDX models of childhood CRLF2-rearranged Ph-like ALL (n=2) and DS-ALL (n=2) and, interestingly, deleterious effects of concomitant JAK inhibition upon CAR T cell functionality. We demonstrated that ruxolitinib co-administration impaired in vivo TSLPRCART-induced ALL cell killing but was also beneficial in protection against life-threatening cytokine release syndrome in co-treated animals. Importantly, TSLPRCART was not eliminated, only suppressed, by JAKi co-treatment with restoration of T cell functionality upon ruxolitinib removal and/or leukemia relapse/rechallenge studies. Ongoing studies are defining optimal TSLPRCART + ruxolitinib sequence(s) to maximize both anti-leukemia efficacy and potential CRS mitigation, as well as assessing in vivo efficacy of bispecific TSLPRCARTs in CRLF2-R Ph-like ALL and DS-ALL PDX models for future translation and clinical evaluation in next-generation trials. Figure 1 Figure 1. Disclosures Fry: ElevateBio: Research Funding; Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Tasian: Aleta Biotherapeutics: Consultancy; Kura Oncology: Consultancy; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding.

Megakaryocyte (MK) development in the bone marrow progresses spatially from the endosteal niche, which promotes MK progenitor proliferation, to the sinusoidal vascular niche, the site of terminal maturation and thrombopoiesis. The chemokine stromal cell-derived factor-1 (SDF-1), signaling through CXCR4, is implicated in the maturational chemotaxis of MKs toward sinusoidal vessels. Here, we demonstrate that both IV administration of SDF-1 and stabilization of endogenous SDF-1 acutely increase MK-vasculature association and thrombopoiesis with no change in MK number. In the setting of radiation injury, we find dynamic fluctuations in marrow SDF-1 distribution that spatially and temporally correlate with variations in MK niche occupancy. Stabilization of altered SDF-1 gradients directly affects MK location. Importantly, these SDF-1-mediated changes have functional consequences for platelet production, as the movement of MKs away from the vasculature decreases circulating platelets, while MK association with the vasculature increases circulating platelets. Finally, we demonstrate that manipulation of SDF-1 gradients can improve radiation-induced thrombocytopenia in a manner additive with earlier TPO treatment. Taken together, our data support the concept that SDF-1 regulates the spatial distribution of MKs in the marrow and consequently circulating platelet numbers. This knowledge of the microenvironmental regulation of the MK lineage could lead to improved therapeutic strategies for thrombocytopenia.

Life-threatening thrombocytopenia can develop following bone marrow injury due to decreased platelet production from megakaryocytes (MKs). However, the study of primary MKs has been complicated by their low frequency in the bone marrow and by technical challenges presented by their unique maturation properties. More accurate and efficient methods for the analysis of in vivo MKs are needed to enhance our understanding of megakaryopoiesis and ultimately develop new therapeutic strategies for thrombocytopenia. Imaging flow cytometry (IFC) combines the morphometric capabilities of microscopy with the high-throughput analyses of flow cytometry (FC). Here, we investigate the application of IFC on the ImageStreamX platform to the analysis of primary MKs isolated from murine bone marrow. Our data highlight and address technical challenges for conventional FC posed by the wide range of cellular size within the MK lineage as well as the shared surface phenotype with abundant platelet progeny. We further demonstrate that IFC can be used to reproducibly and efficiently quantify the frequency of primary murine MKs in the marrow, both at steady-state and in the setting of radiation-induced bone marrow injury, as well as assess their ploidy distribution. The ability to accurately analyze the full spectrum of maturing MKs in the bone marrow now allows for many possible applications of IFC to enhance our understanding of megakaryopoiesis and platelet production. © 2014 International Society for Advancement of Cytometry

Crossing over during meiotic prophase I is required for sexual reproduction in mice and contributes to genome-wide genetic diversity. Here we report on the characterization of an N-ethyl-N-nitrosourea-induced, recessive allele called mei4, which causes sterility in both sexes owing to meiotic defects. In mutant spermatocytes, chromosomes fail to congress properly at the metaphase plate, leading to arrest and apoptosis before the first meiotic division. Mutant oocytes have a similar chromosomal phenotype but in vitro can undergo meiotic divisions and fertilization before arresting. During late meiotic prophase in mei4 mutant males, absence of cyclin dependent kinase 2 and mismatch repair protein association from chromosome cores is correlated with the premature separation of bivalents at diplonema owing to lack of chiasmata. We have identified the causative mutation, a transversion in the 5′ splice donor site of exon 1 in the mouse ortholog of Human Enhancer of Invasion 10 (Hei10; also known as Gm288 in mouse and CCNB1IP1 in human), a putative B-type cyclin E3 ubiquitin ligase. Importantly, orthologs of Hei10 are found exclusively in deuterostomes and not in more ancestral protostomes such as yeast, worms, or flies. The cloning and characterization of the mei4 allele of Hei10 demonstrates a novel link between cell cycle regulation and mismatch repair during prophase I., Author Summary Human infertility and reproductive complications have devastating social and monetary costs. Errors in meiosis during reproduction may lead to birth defects, spontaneous abortion, or infertility. Many of the genes essential for meiosis function in DNA repair and mutations in several of these genes have been shown to contribute to cancer. The identification of the genes necessary for normal meiosis is an important goal and will potentially influence the fields of reproductive and cancer biology. In this study, genetic screens in mice have generated the mutation mei4. mei4 causes male and female sterility by disrupting meiosis and altering the function of the DNA repair system known as mismatch repair. We have identified the causative mutation behind the mei4 phenotype in a gene called Human Enhancer of Invasion 10 or Hei10. This work demonstrates that Hei10 is essential for the completion of meiosis and that it functions to coordinate the DNA repair system and the progression of the cell cycle during meiosis.

The sinusoidal vascular niche is the site of terminal megakaryopoiesis and platelet production in the bone marrow. The association of megakaryocytes (MKs) with the sinusoidal endothelium and microenvironmental cues from the vascular niche are critical for efficient thrombopoiesis. Thrombocytopenia is an important clinical problem that can cause significant morbidity for patients receiving genotoxic treatments such as chemotherapy and radiation therapy. Following myeloablative bone marrow injury, the recovery of both hematopoietic stem and progenitor cells (HSPCs) and the MK lineage is dependent on marrow vascular recovery (Hooper et al. Cell Stem Cell 2009, Avecilla et al. Nature 2004). Accordingly, we reasoned that improving recovery of the vasculature might serve as a rational therapeutic strategy for thrombocytopenia stemming from cytotoxic treatments. We focused our efforts on the arachidonic acid derivative prostaglandin E2 (PGE2), which regulates numerous physiologic processes including microvascular endothelial cell proliferation and angiogenesis. Within the context of the bone marrow, PGE2 also has diverse roles in HSPC expansion, survival, and trafficking. Importantly, systemic administration of the long-acting analogue 16,16-dimethyl PGE2 (dmPGE2) improves hematopoietic recovery and enhances overall survival following total body irradiation (TBI) in mice (Hoggatt et al. Blood Cells 2013). Intriguingly, following sublethal TBI, dmPGE2-induced recovery of peripheral platelets precedes restoration of red blood cells and neutrophils (Porter et al. Stem Cells 2013). We hypothesized that the preferential recovery of platelets following dmPGE2 administration is mediated by accelerated vascular recovery in the marrow. We tested this in a murine model of sublethal radiation injury in which we had previously characterized MK lineage kinetics following 4Gy TBI (Niswander et al. Blood 2014). First, the kinetics of MK progenitors (MKPs), maturing MKs, and platelets were determined at days 5, 7, and 10 in mice receiving 6mg/kg dmPGE2 or vehicle at 48 and 72 hours post-4Gy TBI. Mice receiving dmPGE2 had an early decrease in both MKPs and platelets, with no change in MKs at day 5 post-TBI. However, by day 10, dmPGE2-treated mice demonstrated significantly enhanced recovery of both MKs and circulating platelets compared to controls, but no difference in the number of immunophenotypic HSPCs. Having confirmed enhanced recovery of the MK lineage with dmPGE2 treatment, we next sought to characterize vascular recovery. Marked injury and dilation of the marrow sinusoidal vasculature occurs rapidly following TBI. Immunohistochemistry with morphometric analysis revealed no change in vascular dilation at day 5 post-TBI with dmPGE2 treatment. However, mice receiving dmPGE2 exhibited a more than 25% improvement in vascular dilation that was first evident at day 7 and was sustained at day 10 post-TBI. While vascular dilation post-TBI has been posited to correlate with loss of surrounding hematopoietic cells, improvement in vascular dilation with dmPGE2 treatment is contrastingly evident in the setting of slightly fewer (day 7) or equivalent (day 10) numbers of nucleated marrow cells compared to irradiated control mice. These data suggest that dmPGE2-mediated recovery of the vasculature coincides temporally with the accelerated onset of MK lineage recovery. In addition, immunohistochemical morphometric analyses indicate that dmPGE2-treated mice had an 80% increase in maturing MKs in physical contact with the vascular endothelium, the site of platelet production, at day 10 post-TBI. Taken together, these data support the concept that dmPGE2-mediated marrow vascular recovery following radiation injury enhances the recovery of the MK lineage and accelerates the production of circulating platelets. Modulation of vascular recovery with dmPGE2 may be a promising therapeutic strategy for thrombocytopenia secondary to myeloablative bone marrow injury. Disclosures Calvi: Fate Therapeutics: Patents & Royalties.

The development of megakaryocytes (MKs) in the bone marrow progresses spatially from the endosteal niche, which promotes MK progenitor proliferation, to the sinusoidal vascular niche, the site of terminal maturation and thrombopoiesis. The chemokine SDF-1 (CXCL12), signaling through receptor CXCR4, is produced by stromal cell populations throughout the marrow and is implicated in the maturational chemotaxis of MKs to the sinusoids. Understanding the regulation of MK localization has significance not only for optimal platelet production and the development of therapies for thrombocytopenia, but also in light of the recently proposed role for MKs in supporting hematopoietic stem cells (Heazlewood et al. 2013). In the injury setting of lethal total body irradiation (TBI), it was observed that radioresistant mature MKs relocate to the endosteal niche (Dominici et al. 2009, Olson et al. 2013). Complicating the study of marrow niches post-TBI is the vascular dilation that accompanies the drastic loss of marrow cells. Having confirmed that MKs relocate to the endosteum in our model of sublethal radiation-induced thrombocytopenia (4Gy TBI), we asked whether this localization is due to changes in the spatial distribution of the vasculature or to altered microenvironmental SDF-1. In agreement with other TBI models, we find a significant elevation in SDF-1 transcript levels in the marrow at days 1-3 following 4Gy TBI. Radioresistant MKs, which do not decrease in number until after 3 days, have significantly increased CXCR4 surface expression, a finding we also observe following SDF-1 stimulation of MKs both in vitro and in vivo. In situ hybridization was used to localize the spatial distribution of SDF-1 RNA in femoral marrow. At 2 days post-4Gy, a significant SDF-1 gradient develops with 30% higher SDF-1 message adjacent to the endosteum than in the central marrow. However, this gradient is dynamically eliminated 24 hours later at 3 days post-TBI. These shifts in SDF-1 expression are accompanied by parallel changes in the spatial distribution of MKs by immunohistochemistry. At 2 days post-TBI, there is over a 40% increase in MK in the endosteal niche. In contrast, MKs in the endosteal niche decrease by more than 15% at 3 days, coincident with a significant increase in the MKs associated with vascular endothelium. Thus, these data suggest that the spatial distribution of MKs is dependent upon the localization of SDF-1 in the rapidly fluctuating post-injury bone marrow. To determine if SDF-1 functionally contributes to MK niche changes, we stabilized endogenously-produced SDF-1 using Diprotin A, an inhibitor of SDF-1-inactivating protease DPP4. In uninjured marrow, Diprotin A treatment causes over a 30% rise in MK association with vasculature and a 20% increase in circulating platelets 24 hours later, with no change in MK number. Elevation of vascular SDF-1 by intravenous (IV) administration yields similar results. These data indicate that an endogenous SDF-1 gradient toward the vasculature contributes to homeostatic megakaryopoiesis and thrombopoiesis. At 2 days post-TBI, when endosteal SDF-1 message is increased, stabilization with Diprotin A results in a 40% decrease in MKs associated with vasculature and a small but significant decrease in platelets 24 hours later. Further supporting a role for altered SDF-1 gradients, elevating vascular levels with IV SDF-1 at 2 days causes the opposite effect of Diprotin A, with more MKs found in the vascular niche and a rise in peripheral platelet count. In contrast, at 3 days post-TBI, stabilization of endogenous SDF-1 with Diprotin A causes a further 25% increase in MKs in the vascular niche and a 10% rise in circulating platelets, consistent with the rapid loss of the endosteal SDF-1 gradient. Taken together, our data demonstrate that changes in microenvironmental SDF-1 regulate the spatial distribution of MKs in the post-TBI bone marrow. Importantly, the observed SDF-1 changes have functional consequences for platelet production, as the movement of MKs toward the endosteum decreases circulating platelets, while MK association with the vasculature increases circulating platelets. This knowledge will ultimately lead to improved therapeutic strategies to enhance platelet output in the setting of thrombocytopenia and highlights the need to carefully optimize the timing of therapeutic interventions. Disclosures: No relevant conflicts of interest to declare.

Abstract 2306 Thrombocytopenia complicates many diseases and can be a life-threatening consequence of genotoxic treatments including chemotherapy and radiation therapy. It is well established that thrombopoiesis occurs in the bone marrow where mature megakaryocyte (MK) precursor cells associate with sinusoidal endothelial cells and extrude pro-platelets into the vasculature. There has been much interest in elucidating mechanisms that control megakaryopoiesis and in utilizing these pathways to increase platelet output. The leading paradigm of megakaryopoiesis centers on the ability of cytokines, chiefly thrombopoietin (TPO), to promote MK progenitor proliferation and MK precursor maturation. More recently, attention has been focused on the ability of the bone marrow microenvironment to promote MK maturation and platelet formation. The chemokine stromal-derived factor-1 (SDF-1, also known as CXCL12), signaling through its receptor CXCR4, is implicated in the chemotaxis of MKs toward sinusoidal vessels, and in vivo evidence demonstrates that sustained plasma elevation of SDF-1 can increase platelet counts (Avecilla et al. Nature Medicine, 2004). To more specifically determine the short-term effects of SDF-1, we injected mice with a single 400ng intravenous dose of SDF-1 and enumerated the progenitor, precursor, and platelet compartments of the MK lineage. At 24 hours, SDF-1 induced a 30% increase in platelets compared to vehicle control (p0.7 and p>0.5). To quantitatively determine if SDF-1 regulates the physical interactions of MK precursors with sinusoidal endothelium, we developed a double immunohistochemistry assay using Gp1Bβ to distinguish MK precursors and MECA32 to identify vascular endothelial cells. In vehicle-treated mice, 39% of MKs in the marrow localized to the sinusoidal endothelium, and this increased to 53% 24 hours following SDF-1 treatment (p0.4 and p>0.7). As the platelet response following SDF-1 was less robust in the setting of TBI injury, we hypothesized that SDF-1-induced thrombopoiesis may improve if the number of MK precursors available to move to the vascular niche is increased. To test this, we administered TPO and SDF-1 at 2 hours and at 4 days, respectively, following TBI. TPO treatment alone resulted in 15% more MK precursors than irradiated vehicle controls at 5 days post-TBI (p0.2). In contrast, mice receiving both TPO and SDF-1 had over 20% more platelets than irradiated vehicle controls (p0.9). Correcting for differences in MK precursor numbers, mice receiving TPO and SDF-1 post-TBI had 1.8-fold more MKs in the vascular niche than irradiated vehicle controls (p Disclosures: No relevant conflicts of interest to declare.