Hematopoietic Colony Formation Research Articles

Abstract We082: Contribution of local hematopoiesis to endocardial cushion remodeling during development

Introduction: The cells that comprise the circulatory system not only share their origins but also mutually promote each other’s differentiation during the formation of functional circulatory system. We previously reported a subset of endocardial cells is hemogenic during early embryogenesis. They are enriched in the cushion region, the primordia of the cardiac valves and septa, where active remodeling via endothelial-mesenchymal transition takes place. Hemogenic endocardial cells undergo endocardial-hematopoietic transition (EHT) via Nkx2-5-dependent manner, suggesting that Drosophila tinman-dependent cardio-hematopoietic program is conserved in mammals. Methods: In this study, we analyzed the regulatory network of Nkx2-5-dependent endocardial hematopoiesis using a scRNA-seq data from wildtype and Nkx2-5-null embryonic hearts. In vivo genetic analysis were performed to confirm the candidate signal pathways driven under Nkx2-5 transcription. Results and Conclusion: Nkx2-5-null hearts were devoid of clusters for hemogenic endocardium and cushion endocardium. Interestingly, scRNA-seq analysis further revealed that genes related to Notch signaling pathway are significantly downregulated in Nkx2-5-null endocardium. A further gene network analysis identified that Dhrs3, an enzyme that attenuates retinoic acid (RA) signal by catalyzing the reduction of all-trans-retinaldehyde to all-trans-retinol, is a signature gene of the hemogenic endocardial cells downstream of Nkx2-5. Although RA signal is known to induce the formation of multipotent progenitor, our ex vivo hematopoietic colony forming assay revealed that macrophage formation is strongly inhibited by RA signal. Notch inhibition also suppressed the formation of macrophages. Consistently, in vivo forced activation of NICD drastically increased the number of CD41 + hemogenic endocardial cells as well as macrophages in the cardiac cushion. Strikingly, impaired hematopoiesis and cushion defects in the Nkx2-5-null heart were both rescued by overexpression of NICD, suggesting that Notch signaling promotes endocardial hematopoiesis downstream of Nkx2-5. Taken together, our study demonstrated that the Nkx2-5/RA/Notch signaling axis plays a pivotal role in EHT during early embryogenesis, thereby facilitating local tissue remodeling by inducing macrophage differentiation. The talk will include the latest findings on the specific role of endocardially-derived tissue macrophages.

Abstract 4493: Protein structure inspired drug discovery

Abstract Introduction: Drug action is mediated by a small molecule therapeutic binding to a specific structural motif on a protein. As structure determines function, it follows that unique structural motifs are enriched for sites of unique function and high therapeutic target value. We hypothesized that protein structure constitutes high value primary information that can drive the discovery of novel therapeutic agents. Method: We developed a unique physics-based protein structure analysis platform scaled to run on a supercomputer, allowing us to consider dynamic protein flexibility and the influence of solvent upon protein structure, and to probe large protein libraries as a primary source of structure information that can support up-front structure-based screens. We developed and then probed a high resolution protein x-ray crystallographic library, created and curated by us. From a 60 million compound library, we conducted virtual screens for docking to identified sites. 8-day colony formation assays were conducted on a panel of 8 human breast, prostate, lung and colon cancer cell lines, as well as 8- and 14-day trilineage hematopoietic colony formation assays on human cord blood stem cells. Findings: From the first 180 deposited protein structures, we screened for unique protein surface structure. Structures were deemed unique if they were not otherwise known to be associated with any particular functional roles and were not the binding site of known therapeutics. Further selection included those that were clefts and possessed physical characteristics compatible with binding small chemicals whose properties have been linked to effective therapeutics. For each of 8 sites on 6 different proteins, a suite of analytics probed docking solutions from the compound library, generating a ranked list of 100 compounds for each site. Based on factors inclusive of availability and low potential for toxicity, compounds were acquired and used in in-parallel selection and de-selection functional assays. Cancer cell growth inhibition was a positive selection criterion, with additional ranking based on greater potency and broader efficacy. Bone marrow toxicity is limiting for most drugs, and inhibition of bone marrow stem cell growth was as a deselection criterion. Multiple compounds clustered around 2 sites on 2 different proteins. One compound, Dxr2-017, exhibited very high relative efficacy against colon and breast cancer cells. Evaluation of Dxr2-017 in the NCI-60 panel corroborated these findings, and showed even higher efficacy against melanoma. We demonstrated that Dxr2-017 inhibited melanoma growth with low nanomolar efficacy, while concentrations of over 2300-fold higher had only limited inhibitory effects on bone marrow stem cells. Conclusions: This study provides proof-of-principle that protein structure constitutes high value primary information that can support identification of novel therapeutics. This approach is widely applicable and expandable. Citation Format: Fangfang Qiao, T. Andrew Binkowski, Wayne Anderson, Weining Chen, Gray Schiltz, Karl Scheidt, Amarnath Natarajan, Raymond Bergan. Protein structure inspired drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4493.

A Dual-Specific Inhibitor of Rock/Aurk, RR-1752, for Primary Myelofibrosis

Myelofibrosis (MF) is a clonal myeloproliferative neoplasm (MPN) characterized by driver mutations in JAK2, CALR, and MPL resulting in constitutive activation of JAK-STAT signaling. Abnormalities in megakaryocyte (MK) numbers and morphology are a common feature of MF where abnormal MKs are associated with bone marrow fibrosis and pro-inflammatory cytokine release. Therapies that target constitutive JAK-STAT signaling and atypical megakaryocytes could provide clinical benefit for patients with MF. ROCK and Aurora kinase (AURK) are overexpressed in MPN and linked to dysregulated MKs of patients with MPN. We therefore tested the activity of RR-1752, a dual-specific inhibitor of ROCK and AURK, in preclinical models of MF. RR-1752 acted as an inhibitor of pSTAT3 in IL-2 stimulated human PBMC 1h post stimulation and significantly inhibited phosphorylated myosin light chain 2 (pMLC2) in two human MPN cell lines, HEL-92.1.7 and UKE-1, confirming an on-target action. RR-1752 inhibited colony formation by HEL-92.1.7 and UKE-1, (IC 50 = 25.2 nM and 54.6 nM respectively). RR-1752 also induced cell cycle arrest in HEL-92.1.7 and UKE-1 specifically at the G2/M checkpoint. RR-1752 was also tested in a hematopoietic colony formation assays of MF patient derived CD34 + cells. RR-1752 led to concentration specific inhibition of MF patient derived CD34 + cell colony formation between 100-190 nM demonstrating relevant activity against patient-derived MF samples (51% inhibition at 190 nM as compared to vehicle control). These data were in line with flow cytometrically determined cell viability data of MF CD34 + cells cultured in the presence of varying concentrations of RR-1752 in liquid culture. The in vivo activity of RR-1752, was examined using the human HEL- 92.1.7 cell line, homozygous for both JAK2 V617F and p53 M133K in a mouse xenograft model. The effects of RR-1752 alone and in combination with ruxolitinib were compared to single agent ruxolitinib. The administration of RR-1752, 25 mg/kg PO daily resulted in a significant (p<0.01) improvement in median survival (31 days) compared to ruxolitinib alone, 45 mg/kg PO daily (21 days). RR-1752 5.0 mg/kg PO QD plus ruxolitinib 45 mg/kg PO QD also resulted in a significant improvement in survival (p<0.05) compared to single agent ruxolitinib but was inferior to single agent RR-1752 at 25mg/kg. The demonstrated activity in MF patient derived CD34 + cells combined with the significant improvement in survival compared to the approved drug ruxolitinib in an aggressive, JAK2/p53 mutant MF xenograft model provides a compelling case for its evaluation as a therapeutic modality in MF patients. Dose optimization of RR-1752 alone and in combination with ruxolitinib in the HEL-92.1.7 IV dissemination model as well as testing in the hMPL-W515L-evoked myelofibrosis model is ongoing. Conventional treatments for MF have largely focused on symptom management but RR-1752 treatment could result not only in beneficial symptom improvement but also important disease modifying potential by inhibiting JAK-STAT signaling, cell cycle progression and targeting dysregulated MKs.

Oxidized Mitochondrial DNA Engages TLR9 to Activate the NLRP3 Inflammasome in Myelodysplastic Syndromes.

Myelodysplastic Syndromes (MDSs) are bone marrow (BM) failure malignancies characterized by constitutive innate immune activation, including NLRP3 inflammasome driven pyroptotic cell death. We recently reported that the danger-associated molecular pattern (DAMP) oxidized mitochondrial DNA (ox-mtDNA) is diagnostically increased in MDS plasma although the functional consequences remain poorly defined. We hypothesized that ox-mtDNA is released into the cytosol, upon NLRP3 inflammasome pyroptotic lysis, where it propagates and further enhances the inflammatory cell death feed-forward loop onto healthy tissues. This activation can be mediated via ox-mtDNA engagement of Toll-like receptor 9 (TLR9), an endosomal DNA sensing pattern recognition receptor known to prime and activate the inflammasome propagating the IFN-induced inflammatory response in neighboring healthy hematopoietic stem and progenitor cells (HSPCs), which presents a potentially targetable axis for the reduction in inflammasome activation in MDS. We found that extracellular ox-mtDNA activates the TLR9-MyD88-inflammasome pathway, demonstrated by increased lysosome formation, IRF7 translocation, and interferon-stimulated gene (ISG) production. Extracellular ox-mtDNA also induces TLR9 redistribution in MDS HSPCs to the cell surface. The effects on NLRP3 inflammasome activation were validated by blocking TLR9 activation via chemical inhibition and CRISPR knockout, demonstrating that TLR9 was necessary for ox-mtDNA-mediated inflammasome activation. Conversely, lentiviral overexpression of TLR9 sensitized cells to ox-mtDNA. Lastly, inhibiting TLR9 restored hematopoietic colony formation in MDS BM. We conclude that MDS HSPCs are primed for inflammasome activation via ox-mtDNA released by pyroptotic cells. Blocking the TLR9/ox-mtDNA axis may prove to be a novel therapeutic strategy for MDS.

Abstract PR004: High-density CRISPR screens reveal mechanisms of chromatin regulation of stemness networks in acute myeloid leukemia

Abstract Several leukemia-associated oncoproteins activate transcriptional circuits resembling a stem-like state in acute myeloid leukemia (AML). This activation of “stemness” genes is achieved by enlisting the activity of specialized components of the epigenetic machinery. We sought to comprehensively map epigenetic regulators critical for perpetuating these stemness networks in AML, as they may represent new therapeutic targets. To this end, we used a GFP-reporter knocked into the endogenous locus of the key leukemia oncogene and self-renewal-associated gene MEIS1 and conducted a pooled domain-focused CRISPR screen targeting >600 epigenetic modifiers. Through this screen, we identified and validated multiple members of eight distinct chromatin-modifying complexes that were required for sustaining MEIS1 expression in diverse AML subtypes. These included several novel MEIS1 regulators such as TAF6, LDB1, KAT2A, AFF2, JADE3, casein kinase 2 (CK2), ENY2 and SGF29, in addition to previously characterized regulators such as DOT1L, AF10, ENL and HBO1. A secondary pooled CRISPR screen, coupled with a single-cell transcriptome readout (CROP-seq) revealed that the deletion of several of these MEIS1 activators not only reversed MEIS1 activation but also reduced expression of stem-cell associated genes, including genes of the HOXA cluster, BMI1, SATB1 and Musashi 2, and concomitantly activated expression of differentiation-associated genes. Of particular interest to us was the TUDOR domain chromatin reader SGF29, a key component of the SAGA (Spt-Ada-Gcn5 acetyltransferase) complex. CRISPR-ko of SGF29 significantly reduced the proliferation of cells with distinct HOX-activating mutations including KMT2A rearranged and AF10-rearranged AML cells. ChIP-seq studies showed that SGF29 occupied the promoters and enhancers of key leukemia-oncogenes in a TUDOR-domain-dependent manner and SGF29 deletion selectively attenuated their transcription. Importantly, chromatin proteomics showed that SGF29 deletion led to the eviction of the SAGA complex subunit KAT2A from chromatin and into the cytoplasm. Further, SGF29 knockout impaired blast colony formation and induced differentiation in the KMT2A-MLLLT3, KMT2A-AF10, and CALM-AF10 mouse AML models but did not affect normal hematopoietic colony formation. Lastly, SGF29 deletion delayed disease latency in two distinct human AML cell line models as well as a patient-derived xenograft model in vivo and resulted in striking antiproliferative effects. Our study revealed several new attractive nodes for therapeutic targeting of leukemia stem cells in AML, including the chromatin reader SGF29, and provides a framework to identify vulnerabilities against recalcitrant oncogenic networks. Citation Format: Karina O. Barbosa Guerra, Anagha Deshpande, Ping Xiang, Anna Minkina, Fiorella Schischlik, Adam Brown, Neil A. Robertson, John Doench, Peter D. Adams, Keith Humphries, Eytan Ruppin, Jay Shendure, Prashant Mali, Aniruddha Deshpande. High-density CRISPR screens reveal mechanisms of chromatin regulation of stemness networks in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR004.

Skewed fate and hematopoiesis of CD34+ HSPCs in umbilical cord blood amid the COVID-19 pandemic.

Umbilical cord blood (UCB) is an irreplaceable source for hematopoietic stem progenitor cells (HSPCs). However, the effects of SARS-CoV-2 infection and COVID-19 vaccination on UCB phenotype, specifically the HSPCs therein, are currently unknown. We thus evaluated any effects of SARS-CoV-2 infection and/or COVID-19 vaccination from the mother on the fate and functionalities of HSPCs in the UCB. The numbers and frequencies of HSPCs in the UCB decreased significantly in donors with previous SARS-CoV-2 infection and more so with COVID-19 vaccination via the induction of apoptosis, likely mediated by IFN-γ-dependent pathways. Two independent hematopoiesis assays, a colony forming unit assay and a mouse humanization assay, revealed skewed hematopoiesis of HSPCs obtained from donors delivered from mothers with SARS-CoV-2 infection history. These results indicate that SARS-CoV-2 infection and COVID-19 vaccination impair the functionalities and survivability of HSPCs in the UCB, which would make unprecedented concerns on the future of HSPC-based therapies.

Metformin Inhibits STAT3 and MAPK Signaling through AMPK Activation in CSF3RT618I Cells

Introduction: In the light of the discovery of the CSF3R mutation in chronic neutrophilic leukemia (CNL) patients, the T618I mutation demonstrated to be a molecular marker and a possible target for new therapies due to the JAK/STAT signaling pathway activation [N Engl J Med (2013) 368(19):1781-90]. Since no curative treatment options are available for patients diagnosed with CNL, it is of interest the study new therapeutical approaches for these patients. In this regard, the treatment of JAK2V617F murine models with metformin, an FDA-approved drug for type 2 diabetes, reduced splenomegaly, extramedullary hematopoiesis, and colony formation of HSPC [Cell Death Dis. 2018;9(3):311]. Hence, we characterized the preclinical cellular and molecular effects of metformin in CSF3RT618I models. Methods: The in vitro experiments were performed in Ba/F3 cells transduced retrovirally with CSF3RT618I overexpression (OE) model. Cells were treated with metformin (1-20 mM) to access cell viability and survival. Protein expression was evaluated by western blot. For in vivo evaluation, eight-week-old female NSG mice were transplanted with 2x106 Ba/F3 CSF3RT618I cells, resuspended in Matrigel: PBS (1:1 ratio). After the tumor formation (2 days), mice were treated for 10 days by intraperitoneal injection of vehicle (PBS) or metformin (125 mg/kg, n=10/group). Tumor volume was measured using the formula (V = W2 × L × 0.52), where W and L are the smallest and largest diameters, respectively. Ex vivo primary cells from a patient diagnosed with CNL harboring the CSF3RT618I mutation were used to assess the effects of metformin treatment. The primary bone marrow mononuclear cells were evaluated regarding cell survival and clonogenic capacity upon metformin treatment. All experiments were approved by the Animal and Human Ethics Committee of the Institution. Statistical analyses were performed by Mann-Whitney or ANOVA test and Bonferroni post-test. A p-value < 0.05 was considered. Results: After 24 and 48 hours, Ba/F3 CSF3RT618I treated with 1, 5, 10 and 20 mM of metformin presented a reduction in cell viability in a dose- and time-dependent manner (p < 0.01). The IC50 for metformin treatment for 24, 48, and 72 hours was 17.44, 1.3, and 3.2 mM, respectively. Increased frequency of apoptotic cells was observed at 5, 10, and 20 mM of metformin at times of 24, 48, and 72 hours (p < 0.01). Molecularly, CSF3RT618I cells displayed reduced STAT3 and ERK phosphorylation and increased AKT/mTOR activation, upon metformin 5 mM treatment for 24 hours. AMPK activation was observed in cells treated with 1-20 mM of metformin compared to vehicle. In vivo, metformin treatment decreased the tumor burden of CSF3RT618I transplanted mice after 9 days of treatment (vehicle versus metformin mean ± SEM: 731.9±90.14 vs. 481.6±38.14; p = 0.035). At the protein level, we observed inhibition in Cyclin D1 and the presence of caspase 3 cleavage in the isolated tumor from the metformin-treated mice. Ex vivo metformin treatment of the primary cells isolated from the CNL patient harboring CSF3RT618I resulted in increased apoptosis, with 51.94% (p < 0.05), 52.40% (p < 0.05), and 63.1% (p < 0.001), of apoptosis induction upon treatment with 5, 10 and 20 mM. Metformin ex vivo treatment strongly reduced hematopoietic progenitor cells colony formation on primary CNL CSF3RT618I peripheral blood (83.5% at 10 mM and 100% at 20 mM) and bone marrow mononuclear cells (100% at 10 mM and 98% at 20 mM). Conclusions: We provide evidence that metformin exerts anticancer activity in CSF3RT816I cells, by downregulation of STAT3 and MAPK signaling through AMPK activation. These results support the idea that metformin can be repurposed in the CNL clinics, as a therapeutical option.

Characterisation of canine CD34+/CD45 diminished cells by colony-forming unit assay and transcriptome analysis.

Haematopoietic stem and progenitor cells (HSPCs) are used for transplantation to reconstruct the haematopoietic pathways in humans receiving severe chemotherapy. However, the characteristics of canine HSPCs, such as specific surface antigens and gene expression profiles, are still unclear. This study aimed to characterise the haematopoietic ability and gene expression profiles of canine bone marrow HSPCs in healthy dogs. In this study, the CD34 positive (CD34+) cells were defined as classical HSPCs, CD34+/CD45 diminished (CD45dim) cells as more enriched HSPCs, and whole viable cells as controls. Haematopoietic abilities and gene expression profiles were evaluated using a colony-forming unit assay and RNA-sequencing analysis. Canine CD34+/CD45dim cells exhibited a significantly higher haematopoietic colony formation ability and expressed more similarity in the gene expression profiles to human and mouse HSPCs than those of the other cell fractions. Furthermore, the canine CD34+/CD45dim cells expressed candidate cell surface antigens necessary to define the canine haematopoietic hierarchy roadmap. These results indicate that the canine CD34+/CD45dim cells express the HSPC characteristics more than the other cell fractions, thereby suggesting that these cells have the potential to be used for studying haematopoietic stem cells in dogs.

A Murine Model with JAK2V617F Expression in Both Hematopoietic Cells and Vascular Endothelial Cells Recapitulates the Key Features of Human Myeloproliferative Neoplasm

IntroductionAlthough murine models have provided unequivocal evidence that the JAK2V617F mutation is able to cause myeloproliferative neoplasms (MPNs), there is significant heterogeneity in disease phenotypes between different murine models and none has been able to recapitulate both the myeloproliferative phenotype and cardiovascular pathology seen in patients with MPNs. In addition, these murine models were mostly followed for less than 3-9 months and how aging affects MPN disease progression has not been studied. Endothelial cells (ECs) are an essential component of the hematopoietic niche, and they have been shown to express the JAK2V617F mutation in patients with MPNs. In this study, we investigated how MPN progresses in the JAK2V617F-bearing vascular niche during aging.MethodsJAK2V617F Flip-Flop (FF1) mice (which carry a Cre-inducible human JAK2V617F gene driven by the human JAK2 promoter) were crossed with Tie2-cre mice to express JAK2V617F specifically in all hematopoietic cells (including HSPCs) and vascular ECs (Tie2FF1).ResultsThe Tie2FF1 mice developed essential thrombocythemia to primary myelofibrosisdisease transformation with extramedullary splenic hematopoiesis during 18-month follow up. No evidence of leukemia transformation was observed in the Tie2FF1 mice. (Figure 1)Hematopoietic colony formation assays, flow cytometry analysis, and in vitro culture experiments revealed that there was a loss of both HSPC number and HSPC function in the marrow of old Tie2FF1 mice during aging, mimicking the advanced phases of myelofibrosis. In contrast, the spleen of old Tie2FF1 mice was able to maintain the expansion of JAK2V617F mutant hematopoiesis during aging and MPN disease progression. (Figure 2)These differences between marrow and spleen hematopoiesis in the old Tie2FF1 mice prompted us to investigate how aging affects the JAK2V617F mutant hematopoiesis differently in the marrow and spleen. We found that, although the JAK2V617F mutant HSCs (Lin -cKit +Sca1 +CD150 +CD48 -) from old Tie2FF1 mice were more proliferative than wild-type HSCs in both the marrow and spleen, mutant marrow HSCs were more apoptotic and senescent than wild-type HSCs in the marrow while mutant spleen HSCs were relatively protected in the spleen. Examination of the hematopoietic vascular niche revealed that marrow ECs (CD45 -CD31 +) were significantly decreased in old Tie2FF1 mice compared to age-matched control mice; in contrast, spleen ECs were significantly expanded and less senescent in old Tie2FF1 mice compared to control mice. Therefore, the different vascular niche function of the marrow and spleen could contribute to the decreased marrow hematopoiesis and expanded splenic hematopoiesis we have observed in the Tie2FF1 mice during aging. (Figure 3)Previously, we reported that the Tie2FF1 mice developed spontaneous heart failure with thrombosis, vasculopathy, and cardiomyopathy at 20wk of age. Here, we followed the cardiovascular function of Tie2FF1 mice during aging. At 18mo of age, the Tie2FF1 mice continued to demonstrate a phenotype of dilated cardiomyopathy with a moderate but significant decrease in left ventricular ejection fraction and an increase in left ventricular volume and mass compared to age-matched control mice. Histology examination revealed spontaneous thrombosis in the right ventricle, pulmonary arteries, both main (epicardial) coronary arteries and scattered coronary arterioles (microvessels) in the old Tie2FF1 mice, while age-matched Tie2-cre control mice had no evidence of spontaneous thrombosis in their heart or lungs. Despite these cardiovascular dysfunctions, there was no difference in body weight nor was there any increased incidence of sudden death between the old Tie2FF1 mice and control mice. These findings suggested that there was a persistent but compensated cardiomyopathy and heart failure in the Tie2FF1 mice during aging. (Figure 4)ConclusionCompared to other MPN murine models reported so far, the Tie2FF1 mice is the first MPN murine model that faithfully recapitulated almost all the key features of the human MPN diseases. Considering the presence of the JAK2V617F mutation in microvascular ECs isolated from patients with MPNs and the recapitulation of all the key features of human MPN diseasesby the Tie2FF1 mice, the roles of endothelial dysfunction in the hematologic and cardiovascular pathogenesis of MPN shall be further investigated. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Characterization and Targeting TET2-Mutant HSPCs and AML

Understanding the contribution of abnormal epigenetic programs to acute myeloid leukemia (AML) is necessary for the integrated design of epigenetic-targeted therapies. The protein encoded by TET2 gene is an enzyme that catalyzes the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) as part of the process of DNA demethylation and is frequently mutated in pre-leukemic stem cells. From mice studies, TET2 knockdown/knockout models exhibit a myeloid differentiation bias and an expansion of hematopoietic stem cells (HSCs) with increased myeloproliferation. Notably, no studies to date have specifically examined the cell context effect of TET2 mutations on human hematopoiesis.To understand the mechanism of TET2 mutation-driven leukomogenesis, we have developed an in vitro/ in vivo platform using human CD34+ stem and progenitor cells that mimics TET2-mutated pre-leukemia. Using double chemically modified guide RNAs (gRNAs) coupled with Cas9 recombinant protein, we can disrupt the TET2 gene in cord blood-derived CD34+ cells (average indel frequencies=94.3%, n=6). Beyond disruption, we can modify the TET2 locus through insertion of a GFP cassette using homology driven repair to generate traceable cells. Thus, we have developed a tractable and cell-traceable model that faithfully recapitulates TET2-mutated pre-leukemia and clonal hematopoiesis of indeterminate potential (CHIP).First, we examined the effects of TET2-disruption on human erythroid differentiation in vitro by culturing bulk CD34+GFP+ cells under conditions that promote erythroid differentiation. TET2-disrupted cells produced reduced numbers of glycophorin A/ CD71 double positive erythroid cells compared to AAVS1-targeted control cells. This underscores the importance of TET2 in promoting normal erythroid differentiation, and demonstrates that our methods can give biological effects in a relevant primary human cell system.Next, we investigated the effects of TET2-disruption on hematopoietic colony formation in methylcellulose. Initial colony formation did not differ between TET2-mutant and control cells in the first plating; however, upon the second and third replating, increased numbers of colonies were observed with TET2-disruption. Analysis of indels in the colonies showed that with serial replating, there was enrichment of a 65 base pair deletion that disrupts the entire TET2 locus, suggesting that TET2 knockout cells outcompete normal unmutated hematopoietic stem/progenitor cells (HSPCs) during a month in vitro . We now know that ascorbic acid is an additional co-factor for TET enzymes to hydroxylate 5-mC to 5-hmC. Thus, we also cultured the cells overnight in high dose ascorbic acid prior to plating the cells. In both normoxic and hypoxic conditions, fewer colonies were detected in the TET2 knockout group, suggesting that high dose ascorbic acid cannot restore the TET2 enzymatic function in these cells.Lastly, we conducted in vivo engraftment studies through transplantation of CD34+ cells transfected with TET2-targeting Cas9 RNP (using 2 gRNAs without GFP), and demonstrated successful engraftment of human cells. Four months after transplant, we detected human hematopoietic engraftment with T cells, B cells, and myeloid cells, indicating that Cas9 RNP transfection did not impair engraftment ability. These cells showed high frequencies of indels within the TET2 gene. Surprisingly, we detected a significant proportion of human T cells (approximately 5%, even after transplantation of T cell-depleted CD34+ cells) that were not present with transplantation of control cells. These pilot data demonstrate that Cas9-RNP transfection did not impair engraftment ability of human HSPCs and that TET2-disrupted HSCs can give rise to hematopoietic progeny in vivo with possibly increased production of T cells. Secondary transplants are ongoing to assess for self-renewal.Here, we report the development of a CRISPR/Cas9 approach to target the TET2 gene in primary human HSPCs. We show that TET2 disruption leads to a block in erythroid differentiation, increased colony formation and replating in vitro, and increased engraftment in vivo . This model will enable further studies of effects on the epigenome through DNA methylation and hydroxymethylation studies, as well as provide an in vivo model of pre-leukemia/CHIP associated with TET2 mutations. DisclosuresNo relevant conflicts of interest to declare.

Combination Treatment with an MDM2 Antagonist and a BET Inhibitor Targets Both Myelofibrosis Hematopoietic Stem/ Progenitor Cells and Their Tumor Promoting Microenvironment

Introduction: Myelofibrosis (MF) arises at the level of hematopoietic stem and progenitor cells (HSC/HPC) due to a sequence of mutational events that results in increased JAK/STAT signaling. JAK2 has served as a target for drug development and has led to the development of several novel small molecule inhibitors. Clinical trials of these agents have revealed their ability to decrease the degree of splenomegaly and to improve systemic symptoms of PMF patients, but not to substantially affect the progeny of the malignant clone or the rate of disease progression. MF HSC/HPCs and their progeny are known to elaborate a number pro-inflammatory mediators including lipocalin (LCN2) (Lu et al, Blood, 2015), and IL-8 which are capable of creating a tumor promoting microenvironment. LCN2 promotes osteoblast formation and marrow fibrosis (Lu et al, Blood, 2013) while IL8 promotes endothelial cell proliferation. We have previously reported that plasma LCN2 levels are increased in MF patients (Lu et al, ASH, 2016) and show now that plasma IL-8 levels are also dramatically increased (Figure 1). Furthermore the levels of IL-8 as shown by IHC were increased in splenic myeloid cells especially megakaryocytes and the IL-8 receptor CXCR2 was expressed at greater levels by MF splenic littoral cells that line the sinusoids as well as endothelial cells as compared to normal spleens. We hypothesize that effective treatment of MF would require a combination of drugs that directly deplete the malignant HSC/HPCs as well as dampen the tumor promoting microenvironment. In a previous study, we have found the MDM2 protein, a negative regulator of p53, are highly expressed in PV CD34+ cells from PV patients and can be targeted by nutlin therapy (Lu et al, Blood, 2013). In order to assess if nutlin therapy would deplete MF CD34+ cells, using flow cytometry method, we documented that MDM2 protein expression was significantly increased in MF CD34+ cells as compared to normal CD34+ cells and was higher than that in PV CD34+ cells (Figure 2). This finding suggests that MF malignant HSC/HPC might also be sensitive to the treatment with a nutlin. Furthermore, MF HSC/HPCs reside in the BM and extra medullary sites containing dysfunctional microenvironments which promote the predominance of the malignant stem cells. We assume that BET inhibitors might dampen the MF inflammatory milieu since BET proteins play a central role in regulation of inflammatory responses. BET proteins interact with transcription factors (TF) including NF-KB, P53 and c-Myc, and downregulate the transcriptional activation of numerous cytokines including IL-6 and IL-8. We then hypothesize that treatment with low doses of nutlin combined with JQ1, a BET inhibitor, will target MF malignant cells as well as correct MF HSC tissue specific supportive microenvironments by decreasing pro-inflammatory cytokine generation.Results: 1. Treatment with a combination of low dose RG7112 and JQ1 selectively decreased hematopoietic colony formation by MF CD34+ cells. Exposure to either low dose RG7112 (200nM) or JQ1 (50nM) alone for 2 days increased the degree of MF CD34+ cell apoptosis modestly, while a combination of the 2 drugs significantly induced MF CD34+ cell apoptosis (p<0.05). By contrast, treatment with each drug alone or in combination however did not significant induce apoptosis of normal CD34+ cells. Combination treatment of RG7112 and JQ1 inhibited total number of colonies generated by MF CD34+ cells by 60%, as compared to the treatment with each drug alone (p=0.004 and p=0.03, respectively). Combination treatment with RG7112 and JQ1 did not, however, decrease colony formation by normal HPC. In a small number of cases, combination therapy decreased the numbers of homozygous JAK2V617F+ colonies assayed from MF CD34+ cells (at range from 8% to 20%).2. Both RG7112 and JQ1 treatment decrease the elaboration of MF pro-inflammatory cytokines. Treatment of MF mononuclear cells (MNC) with RG7112 but not JQ1 decreased LCN2 levels in conditioned medium (CM) (at range from 5% to 39%, p=0.012); while treatment with JQ1 at 50nM strongly decreased the levels of IL-8 in MF MNC CM by 50% (p=0.0003).Summary: These data indicate that JQ1 combined with RG7112 might be a potential effective strategy for the treatment of MF. This drug combination will not only selectively target MF HSC/HPCs but also alter the MF HSC promoting microenvironment by inhibiting the elaboration of pro-inflammatory mediators. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A drug targeting 5-lipoxygenase enhances the activity of a JAK2 inhibitor in CD34+ bone marrow cells from patients with JAK2V617F-positive polycythemia vera in vitro.

Janus kinase 2 (JAK2) inhibitors, the first targeted treatments for myeloproliferative neoplasms (MPNs), provide substantial benefits, including a marked reduction in splenomegaly and MPN-associated symptoms. However, these drugs rarely induce molecular remission in patients with MPNs. Zileuton, a 5-lipoxygenase (5-LO) inhibitor, has been demonstrated to selectively deplete hematopoietic stem cells (HSCs) expressing a JAK2 point mutation (JAK2V617F) in mouse models of JAK2V617F-induced polycythemia vera (PV). To determine the potential activity of 5-LO inhibitors in combination with JAK inhibitors against human PV HSCs, the present study first analyzed 5-LO expression in CD34+ bone marrow cells from patients with JAK2V617F-positive PV using western blotting and reverse transcription-quantitative PCR, and then examined the effect of zileuton combined with ruxolitinib on colony formation using a colony formation assay. Furthermore, cell cycle and apoptosis in CD34+ cells from patients with PV and healthy volunteers were determined by flow cytometry. In the present study, 5-LO expression was upregulated in CD34+ cells from patients with PV compared with in CD34+ cells from healthy volunteers. Higher levels of leukotriene B4, a product of the 5-LO signaling pathway, were detected in patients with PV compared with in healthy volunteers. Zileuton treatment suppressed the colony formation of CD34+ cells from patients with PV in a dose-dependent manner. Furthermore, zileuton and ruxolitinib exerted their anticancer effects by suppressing hematopoietic colony formation, inducing apoptosis and arresting the cell cycle of human CD34+ cells from patients with PV. The combination of these two drugs exerted a more beneficial effect than either agent alone. Based on these data, zileuton enhanced the antitumor activity of low-dose ruxolitinib in hematopoietic progenitor cells from patients with PV, providing conceptual validation for further clinical applications of combination treatment with ruxolitinib and zileuton for patients with PV.

Proteoglycans regulate protein tyrosine phosphatase receptor σ organization on hematopoietic stem/progenitor cells

Protein tyrosine phosphatase receptor σ (PTPσ) is highly expressed by murine and human hematopoietic stem cells (HSCs) and negatively regulates HSC self-renewal and regeneration. Previous studies of the nervous system suggest that heparan sulfate proteoglycans can inactivate PTPσ by clustering PTPσ receptors on neurons, but this finding has yet to be visually verified with adequate resolution. Here, we sought to visualize and quantify how heparan sulfate proteoglycans regulate the organization and activation of PTPσ in hematopoietic stem/progenitor cells (HSPCs). Our study illustrates that syndecan-2 promotes PTPσ clustering, which sustains phospho-tyrosine and phospho-ezrin levels in association with augmentation of hematopoietic colony formation. Strategies that promote clustering of PTPσ on HSPCs may serve to powerfully augment hematopoietic function.

Simultaneous Activation of Signals Through gp130, c-kit, and Interleukin-3 Receptor Promotes a Trilineage Blood Cell Production in the Absence of Terminally Acting Lineage-Specific Factors

AbstractWe assessed the biologic role of signaling through gp130, a signal-transducing receptor (R) component, in human hematopoiesis in vitro. Although peripheral blood-derived CD34+ cells ubiquitously expressed gp130 and interleukin-3 receptor α (IL-3Rα), IL-6Rα was only detected on 80% of these CD34+ cells. We sorted CD34+IL-6R+ or CD34+IL-6R− cells and studied the effect on hematopoietic colony formation of signaling through gp130 activated by IL-6 or a combination of IL-6 and recombinant soluble human IL-6R (sIL-6R) in the presence or absence of stem cell factor (SCF ) and/or IL-3. Signals activated by SCF, IL-6, or IL-6/sIL-6R complex alone did not induce significant colony formation. However, a combination of IL-3, SCF, and IL-6/sIL-6R complex dramatically induced many neutrophil (colony-forming unit-granulocyte [CFU-G]), erythroid burst (burst-forming unit-erythrocyte [BFU-E]), erythrocyte-containing mixed (CFU-Mix), and megakaryocyte (CFU-Meg) colony formations when CD34+IL-6R− cells were used as the target. CFU-G colony formation induced by the three signals was more evident when CD34+IL-6R+ cells were used as the target. This distinct synergistic effect of the three different signals was confirmed by single-cell clone-sorting experiments. Moreover, colony formation (including CFU-G, BFU-E, CFU-Mix, and CFU-Meg) was observed even in the presence of neutralizing antibodies for granulocyte colony-stimulating factor, erythropoietin, and thrombopoietin (c-Mpl), whereas neutralizing antibodies for gp130, IL-6R, IL-3, and SCF partially or completely blocked the synergistic effect. The maturation of neutrophilic, erythroid, and megakaryocytic cells supported by the three signals in serum-free cultures was confirmed by immunostaining using anti-CD66b, antiglycophorin A, antihemoglobin α, and anti-CD41 monoclonal antibodies, respectively. In contrast, any two of the three signals were insufficient for effective blood cell production in the absence of maturation factors. These results suggest that simultaneous activation of the three signals through gp130, c-kit, and IL-3R can induce in vitro proliferation and differentiation of trilineage hematopoietic progenitors in the absence of terminally acting lineage-specific factors.

A 3D construct based on mesenchymal stromal cells, collagen microspheres and plasma clot supports the survival, proliferation and differentiation of hematopoietic cells in vivo.

The hematopoietic niche is a specialized microenvironment that supports the survival, proliferation and differentiation of hematopoietic stem progenitor cells (HSPCs). Three-dimensional (3D) models mimicking hematopoiesis might allow in vitro and in vivo studies of the hematopoietic (HP) process. Here, we investigate the capacity of a 3D construct based on non-adherent murine bone marrow mononuclear cells (NA-BMMNCs), mesenchymal stromal cells (MSCs) and collagen microspheres (CMs), all embedded into plasma clot (PC) to support in vitro and in vivo hematopoiesis. Confocal analysis of the 3D hematopoietic construct (3D-HPC), cultured for 24h, showed MSC lining the CM and the NA-BMMNCs closely associated with MSC. In vivo hematopoiesis was examined in 3D-HPC subcutaneously implanted in mice and harvested at different intervals. Hematopoiesis in the 3D-HPC was evaluated by histology, cell morphology, flow cytometry, confocal microscopy and hematopoietic colony formation assay. 3D-HPC implants were integrated and vascularized in the host tissue, after 3months of implantation. Histological studies showed the presence of hematopoietic tissue with the presence of mature blood cells. Cells from 3D-HPC showed viability greater than 90%, expressed HSPCs markers, and formed hematopoietic colonies, in vitro. Confocal microscopy studies showed that MSCs adhered to the CM and NA-BMMNCs were scattered across the 3D-HPC area and in close association with MSC. In conclusion, the 3D-HPC mimics a hematopoietic niche supporting the survival, proliferation and differentiation of HSPCs, in vivo. 3D-HPC may allow evaluation of regulatory mechanisms involved in hematopoiesis.

Generation of hematopoietic stem/progenitor cells with sickle cell mutation from induced pluripotent stem cell in serum-free system.

IntroductionSickle cell disease (SCD) is a monogenic disease and it is estimated that 300,000 infants are born annually with it. Most treatments available are only palliative, whereas the allogeneic hematopoietic stem cell transplantation offers the only potential cure for SCD.ObjectiveGeneration of human autologous cells, when coupled with induced pluripotent stem cell (iPSC) technology, is a promising approach for developing study models. In this study, we provide a simple and efficient model for generating hematopoietic cells using iPSCs derived from a sickle cell anemia patient and an inexpensive in-house-prepared medium.MethodThis study used iPSCs previously generated from peripheral blood mononuclear cells (PBMCs) from a patient with sickle cell anemia (iPSC_scd). Hematopoietic and erythroid differentiation was performed in two steps. Firstly, with the induction of hematopoietic differentiation through embryoid body formation, we evaluated the efficiency of two serum-free media; and secondly, the induction of hematopoietic stem/progenitor cells to erythroid progenitor cells was performed.ResultsThe patient-specific cell line generated CD34+/CD45+ and CD45+/CD43+ hematopoietic stem/progenitor cells and erythroid progenitors, comprising CD36+, CD71+ and CD235a+ populations, as well as the formation of hematopoietic colonies, including erythroid colonies, in culture in a semi-solid medium.ConclusionIn conjunction, our results described a simple serum-free platform to differentiate human the iPSCs into hematopoietic progenitor cells. This platform is an emerging application of iPSCs in vitro disease modeling, which can significantly improve the search for new pharmacological drugs for sickle cell disease.

Injectable, scalable 3D tissue-engineered model of marrow hematopoiesis

Modeling the interaction between the supportive stroma and the hematopoietic stem and progenitor cells (HSPC) is of high interest in the regeneration of the bone marrow niche in blood disorders. In this work, we present an injectable co-culture system to study this interaction in a coherent in vitro culture and in vivo transplantation model. We assemble a 3D hematopoietic niche in vitro by co-culture of supportive OP9 mesenchymal cells and HSPCs in porous, chemically defined collagen-coated carboxymethylcellulose microscaffolds (CCMs). Flow cytometry and hematopoietic colony forming assays demonstrate the stromal supportive capacity for in vitro hematopoiesis in the absence of exogenous cytokines. After in vitro culture, we recover a paste-like living injectable niche biomaterial from CCM co-cultures by controlled, partial dehydration. Cell viability and the association between stroma and HSPCs are maintained in this process. After subcutaneous injection of this living artificial niche in vivo, we find maintenance of stromal and hematopoietic populations over 12 weeks in immunodeficient mice. Indeed, vascularization is enhanced in the presence of HSPCs. Our approach provides a minimalistic, scalable, biomimetic in vitro model of hematopoiesis in a microcarrier format that preserves the HSPC progenitor function, while being injectable in vivo without disrupting the cell-cell interactions established in vitro.

HCMV miR-US22 down-regulation of EGR-1 regulates CD34+ hematopoietic progenitor cell proliferation and viral reactivation.

Reactivation of latent Human Cytomegalovirus (HCMV) in CD34+ hematopoietic progenitor cells (HPCs) is closely linked to hematopoiesis. Viral latency requires maintenance of the progenitor cell quiescence, while reactivation initiates following mobilization of HPCs to the periphery and differentiation into CD14+ macrophages. Early growth response gene 1 (EGR-1) is a transcription factor activated by Epidermal growth factor receptor (EGFR) signaling that is essential for the maintenance of CD34+ HPC self-renewal in the bone marrow niche. Down-regulation of EGR-1 results in mobilization and differentiation of CD34+ HPC from the bone marrow to the periphery. In the current study we demonstrate that the transcription factor EGR-1 is directly targeted for down-regulation by HCMV miR-US22 that results in decreased proliferation of CD34+ HPCs and a decrease in total hematopoietic colony formation. We also show that an HCMV miR-US22 mutant fails to reactivate in CD34+ HPCs, indicating that expression of EGR-1 inhibits viral reactivation. Since EGR-1 promotes CD34+ HPC self-renewal in the bone marrow niche, HCMV miR-US22 down-regulation of EGR-1 is a necessary step to block HPC self-renewal and proliferation to induce a cellular differentiation pathway necessary to promote reactivation of virus.

Elucidating an Essential Role of Promoting Endothelial-to-Hematopoietic Transition By a Key EMT-Regulator ZEB2 Using a Human iPSC-Based Model

ZEB2 (Zinc finger E-box binding homeobox 2; also known as SMAD interacting protein-1 or SIP1) is a DNA-binding transcriptional regulator associated with epithelial-to-mesenchymal-transition (EMT). It has been reported that it is important in many other developmental processes, including mesoderm formation during gastrulation and neural crest formation. More recently, a study reported that inactivation of ZEB2 in mice results in differentiation defects in multiple hematopoietic cell lineages (1). In order to examine the roles of ZEB2 in human developmental process and hematopoiesis, we decided to apply human induced pluripotent stem cells (iPSCs) as a model with a pair of isogenic iPSCs with or without functional ZEB2 expression. We used a validated iPSC-based hematopoietic differentiation under a feeder- and xeno-free culture condition was used to study the role of ZEB2 in hematopoietic cell formation and to avoid other EMT-inducing factors in serum. We found that ZEB2 expression is abundant in either cord blood cells or human iPSCs-derived hematopoietic stem/progenitor cells (HSPCs), indicating that ZEB2 deficiency may hinder human hematopoietic differentiation. To knock out the ZEB2 gene and generate ZEB2 deficient human iPSCs, we used two different CRISPR guide RNAs as well as SpCas9 in a human iPSC line BC1. An engineered isogenic BC1 iPSC line that has both alleles 155-bp deletion in ZEB2 exon 1 (ZEB2 -/-) was successfully isolated and expanded, resulting in the frameshift and the consequent disappearance of ZEB2 protein expression. The complete ZEB2 knockout had little effects on human iPSCs expansion. However, we observed that the differentiation of ZEB-/- BC1 into CD34+CD45+ HSPCs was decreased. The number of ZEB-/- BC1-derived HSPCs was 5-fold lower than that from wide type BC1 control. Moreover, ZEB2-deleted HSPCs were defected in hematopoietic colony formation. To figure out at which hematopoietic differentiating stage the defects occurred, we applied inducible iPSC's endothelium-hematopoietic transformation (EHT) system to mimic hemogenic endothelial cells (ECs) giving rise to HSPCs. By using this established EHT system, ZEB2 deficient iPSCs were differentiated in order into CD34+CD31+CD144+ ECs, and then to HSPCs (2). We found that ZEB2 depletion did not adversely affect the formation of ECs from human iPSCs in this EHT experimental system but did impact on HSPCs generation from ECs. This study is unique in several ways to elucidate the roles of ZEB2 in human hematopoietic development. This prospective genetic model allowed us to pinpoint ZEB2 is critical at the stage of EHT during human hematopoietic cell formation. Our results indicate that contribution of ZEB2 deficiency in blood disorders might result from the inhibition of HSPC formation. The human iPSC-based hematopoietic differentiation system coupled with precisely edited isogenic iPSC lines also provide a more controlled genetic model for studying functions of others gene in their regulation of human hematopoiesis.

The CDK6 Inhibitor Palbociclib in Combination with Ruxolitinib Remarkably Improves Myelofibrosis in Murine Models

Myelofibrosis (MF) is the deadliest form of myeloproliferative neoplasms (MPN), characterized by deposition of fibrous tissues in the bone marrow, abnormal megakaryopoiesis, ineffective erythropoiesis and extramedullary hematopoiesis. The median survival for patients with MF is ~5 years. The JAK2V617F mutation, which results in constitutive activation of the JAK2 tyrosine kinase, has been found in 50-60% patients with MF. Additional mutations in the genes encoding thrombopoietin receptor (MPL) and calreticulin (CALR) have been found at a lower frequency than JAK2V617F in MF. The JAK1/JAK2 inhibitor ruxolitinib has been approved for treatment of MF. Although ruxolitinib can reduce splenomegaly and constitutional symptoms, it does not cure or prevent fibrosis in patients with MPN/MF. Additionally, ruxolitinib treatment does not improve survival in MF patients. So, there is a critical need to identify new therapeutic targets and develop novel therapies for MF. We have found that expression of CDK6 is significantly elevated in mouse and human MPN hematopoietic cells/progenitors. We also observed that knockdown of CDK6 significantly inhibited proliferation of BA/F3-EpoR-JAK2V617F and HEL cells expressing JAK2V617F but not wild-type JAK2 expressing BA/F3-EpoR cells, suggesting that CDK6 is a bona fide target in MPN. In this study, we investigated the efficacy of CDK4/6 inhibitor Palbociclib (PD-0332991) in hematopoietic cells expressing JAK2V617F and MPLW515L and murine models of MF. Since Palbociclib is an FDA-approved drug for treatment of breast cancer, information on the dose, pharmacokinetics and toxicity of Palbociclib in human is already available. Thus, it would be easier to move this drug into clinic and repurpose it for treatment of MPN/MF. We observed that Palbociclib treatment (0.25-0.5 uM) significantly reduced the proliferation of murine BA/F3 cells expressing JAK2V617F or MPLW515L. Palbociclib treatment also significantly inhibited human JAK2V617F-positive HEL, SET-2 and UKE-1 cells. However, Palbociclibonly modestly affected proliferation of wild type JAK2-expressing BA/F3 cells at higher concentrations. Palbociclib treatment also resulted in marked apoptosis in BA/F3-EpoR cells expressing JAK2V617F. Furthermore, Palbociclib and ruxolitinib synergistically induced apoptosis in JAK2V617F-expressing hematopoietic cells. Additionally, Palbociclib significantly inhibited hematopoietic progenitor colony formation in MPN/MF CD34+ cells. We previously generated conditional JAK2V617F knock-in mice. Whereas expression of heterozygous JAK2V617F induces a PV-like disease, homozygous JAK2V617F expression promotes rapid progression to myelofibrosis. We have utilized the homozygous JAK2V617F mice to test the in vivo efficacy of Palbociclib alone and in combination with ruxolitinib against MF. Treatment of Palbociclib alone significantly reduced the WBC and neutrophil counts and decreased the spleen size in homozygous JAK2V617F mice. Combined treatment of Palbociclib and ruxolitinib almost completely normalized the WBC and neutrophil counts and the spleen size in homozygous JAK2V617F mice. Histopathologic analysis revealed marked reduction in fibrosis in the bone marrow of homozygous JAK2V617F mice treated with Palbociclib or Palbociclib and ruxolitinib combination. We also have tested the in vivo efficacy of Palbociclib and Palbociclib/ruxolitinib combination in MPL mutant (MPLW515L) mouse model of MF. We observed similar decrease in WBC and neutrophil counts and reduction in splenomegaly in MPL mutant mice upon treatment of Palbociclib alone or in combination with ruxolitinib. Combined treatment of Palbociclib and ruxolitinib remarkably reduced fibrosis in the bone marrow of MPL mutant mice. Overall, our results suggest that palbociclib in combination with ruxolitinib may have therapeutic potential against MF and support the clinical investigation of palbociclib and ruxolitinib combination in patients with MF. Disclosures No relevant conflicts of interest to declare.