Megakaryocyte Colony Formation Research Articles

Absence of Nkx2-3 induces ectopic lymphatic endothelial differentiation associated with impaired extramedullary stress hematopoiesis in the spleen.

The red and white pulps as two main parts of the spleen are arranged around distinct types of vasculature, and perform significantly different functions in both humans and mice. Previous observations indicated a profound alteration of the local vessel specialization in mice lacking Nkx2-3 homeodomain transcription factor, including contradictory results suggesting presence of an ectopic lymphatic vascular structure. Furthermore, how the absence of Nkx2-3 and the consequential changes in endothelial components affect the extramedullary hematopoietic activity restricted to the splenic red pulp is unknown. In this work, we investigated the role of Nkx2-3 homeodomain transcription factor as a major morphogenic determinant for vascular specification, and its effect in the extramedullary hematopoiesis following acute blood loss and pharmacological stimulation of megakaryocyte differentiation after treatment with thrombopoietin-receptor mimetic Romiplostim. We found that, in mice lacking Nkx2-3, Prox1-positive lymphatic capillaries containing gp38/CD31 double positive lymphatic endothelial cells develop, arranged into an extensive meshwork, while the Clever1-positive venous segments of red pulp blood vasculature are absent. This lymphatic endothelial shift is coupled with a severely compromised splenic erythropoiesis and a significantly reduced splenic megakaryocyte colony formation following Romiplostim treatment in mice lacking Nkx2-3. These findings indicate that the shift of microvascular patterning in the absence of Nkx2-3 includes the emergence of ectopic Prox1-positive lymphatic vessels, and that this pivoting towards lymph node-like vascular patterning is associated with an impaired reserve hematopoietic capacity of the splenic red pulp.

Differentiation therapy for murine myelofibrosis model with MLN8237 loaded low-density lipoproteins.

Primary myelofibrosis (PMF) is a severe myeloproliferative neoplasm that is characterized by low-differentiation megakaryoblasts and progressive bone marrow fibrosis. Although an Aurora kinase A (AURKA) targeting small-molecule inhibitor MLN8237 has been approved in clinical trials for differentiation therapy of high-risk PMF patients, its off-target side effects lead to a partial remission and serious complications. Here, we report a dual-targeting therapy agent (rLDL-MLN) with great clinical translation potential for differentiation therapy of PMF disease. In particular, the reconstituted low-density lipoprotein (rLDL) nanocarrier and the loaded MLN8237 can actively target malignant hematopoietic stem/progenitor cells (HSPCs) via LDL receptors and intracellular AURKA, respectively. In contrast to free MLN8237, rLDL-MLN effectively prohibits the proliferation of PMF cell lines and abnormal HSPCs and significantly induces their differentiation, as well as prevents the formation of erythrocyte and megakaryocyte colonies from abnormal HSPCs. Surprisingly, even at a 1500-fold lower dosage (0.01mg/kg) than that of free MLN8237, rLDL-MLN still exhibits a much more effective therapeutic effect, with the PMF mice almost clear of blast cells. More importantly, rLDL-MLN promotes hematological recovery without any toxic side effects at the effective dosage, holding great promise in the targeted differentiation therapy of PMF patients.

Preliminary Data from the Phase I/II Study of TP-3654, a Selective Oral PIM1 Kinase Inhibitor, in Patients with Myelofibrosis Previously Treated with or Ineligible for JAK Inhibitor Therapy

Introduction: Myelofibrosis (MF) is characterized by bone marrow (BM) fibrosis, ineffective hematopoiesis, splenomegaly, and debilitating symptoms. Expression of PIM-1 is significantly upregulated in MF hematopoietic cells, supporting exploration of PIM-1 as a potential therapeutic target in MF. TP-3654 is a highly selective oral investigational PIM-1 kinase inhibitor. When administered alone and in combination with ruxolitinib, TP-3654 reduced spleen size and BM fibrosis in JAK2V617F and MPLW515L murine MF models (Dutta, 2021). In addition, TP-3654 decreased cytokine response genes and serum TGF-b in progenitor cells and JAK2V617F mice, respectively. TP-3654 showed less hematopoietic inhibition than Janus kinase (JAK) inhibitors (ruxolitinib, pacritinib and momelotinib) in in vitro human megakaryocyte and erythrocyte cell colony formation. Methods: This Phase I/II study evaluates the safety and efficacy of TP-3654 monotherapy in patients with MF (NCT04176198). Key eligibility criteria include primary or secondary MF; intermediate or high-risk MF per DIPSS; previously treated with or ineligible for JAK inhibitor treatment; grade ≥2 BM fibrosis; platelet count ≥25x109/L; absolute neutrophil count ≥1x109/L; splenomegaly; and ≥2 measurable symptoms. The study aims to identify the maximum tolerated dose and/or recommended Phase II dose using a Bayesian logistic regression model (BLRM) with overdose control, and to assess clinical activity (spleen volume reduction [SVR], total symptom score [TSS] improvement, and BM fibrosis reduction), safety, pharmacokinetic, and pharmacodynamic markers in peripheral blood and BM biopsies. Results: As of 11 July 2022, 8 patients were enrolled across 5 dose levels in the dose escalation phase. At baseline, median age was 70 years (range, 61 to 77), median spleen volume was 2370 cm3 (range, 1189 to 4407), median total symptom score was 19 (range, 4 to 62), median platelet count was 120 x109/L (range 68 to 237), median hemoglobin was 10.1 g/dL (range, 5.9 to 13.7), and two patients were transfusion-dependent. All patients had received prior treatment with at least one JAK inhibitor. Median duration of prior JAK inhibitor treatment was 35 weeks (range, 13 to 269). Six patients had JAK2V16F mutation, 2 patients had CALR mutation. The median duration of TP-3654 treatment was 21 weeks (range, 1 to 45). No dose-limiting toxicities occurred. Treatment-related adverse events (TRAEs) occurring in >20% of patients included mild to moderate nausea, vomiting, and diarrhea. Grade ≥ 3 TRAEs included only 1 case of vomiting. No hematological TRAEs were reported. No discontinuation due to AE occurred. SVR was observed in 5 out of 6 evaluable patients (median best change -14%, range -3.2% to -35%) (Figure 1). Symptom improvement was observed in 5 out of 6 evaluable patients (median best change -70%, range -32 to -100%). A panel of cytokines were evaluated in plasma samples obtained at baseline and during TP-3654 treatment. Reductions in cytokines (TGF-b, IL-18, VEGF, RANTES, MMP-9, and TIMP-1) were observed after TP-3654 treatment (Figure 2). Patients with higher cytokine reductions correlated with higher reduction in total symptom score. Enrollment is ongoing, updated data will be presented. Conclusions: The preliminary clinical data in dose escalation show: 1) encouraging signs of clinical activity in spleen volume reduction, symptom improvement, and cytokine reduction with TP-3654 monotherapy in patients previously treated with JAK inhibitors, 2) TP-3654 is well tolerated with limited myelosuppressive adverse events. The non-clinical findings and preliminary clinical safety/efficacy data support accelerated development and assessment of TP-3654 as the optimal partner for combination with JAK inhibitors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Introduction and Genetic Correction of Calreticulin Mutations in Human Hematopoietic Stem and Progenitor Cells Sheds Light on MPN Pathogenesis

Introduction:Recurrent mutations in calreticulin (CALR) are present in 70% to 80% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients without a JAK2 or MPL mutation. Despite recent advances in understanding mutant CALR, the detailed mechanisms are not fully elucidated, and current knowledge is mainly based on transgenic mouse models or human cancer cell lines. Thus, to more faithfully model MPN pathogenesis, we first aimed to introduce heterozygous type-1 and type-2 CALR mutations into healthy human hematopoietic stem and progenitor cells (HSPCs) via targeted CRISPR/Cas9-mediated gene knock-in (KI) and investigate its impact on HSPC function in vitro and in vivo. Second, we aimed to correct CALR mutations in patient-derived HSPCs to study their dependence on the initial driver event to exert an MPN phenotype.Methods:We used CRISPR/Cas9 to introduce heterozygous CALR mutations into the endogenous gene locus of healthy cord blood-derived HSPCs. Our approach is based on homologous recombination using DNA repair templates delivered by adeno-associated virus serotype 6 (AAV6). Briefly, Cas9-sgRNA ribonucleoprotein (RNP) was used to cut the DNA. Simultaneously AAV6, carrying either a mutation-bearing or a wildtype control cDNA, was co-delivered to allow for targeted in-frame integration. This way, mutant CALR remains under the control of the endogenous promoter. Concurrent integration of a fluorescent reporter downstream of the mutated exon, enabled purification and tracking of modified cells via flow cytometry. Purified CRISPR-modified HSPCs were used for in vitro collagen-based colony-forming assays, proliferation and differentiation assays in liquid culture, and intrafemoral transplantation into immunodeficient NSG mice to assess their pathogenic potential.Results:Our CRISPR/Cas9 KI strategy enabled us to efficiently generate and enrich for heterozygous CALR mutant human HSPCs. Modified cells harbor the mutation at the endogenous CALR locus with intact gene regulatory regions. Correct integration and transcript expression were confirmed on DNA and RNA level by sanger sequencing. Additionally, CALR mutant protein expression was confirmed via immunohistochemistry using a diagnostically approved mutant-specific antibody.Type-1 and type-2 CALR mutations led to TPO-independent growth of CD34 + HSPC-derived cells and a two-fold (p<0.01) increase of megakaryocyte colonies in collagen-based media compared to wildtype control KI. These findings were corroborated by significantly enhanced CD41 + CD42b + megakaryocyte formation of CALR mutant HSPCs upon liquid culture differentiation.When transplanted into sublethally irradiated immunodeficient NSG mice, CALR mutant HSPCs showed robust engraftment in the bone marrow with a myeloid lineage skewing, outcompetition of wildtype cells and increased formation of CALR mutant CD41 + megakaryocyte progenitors.To investigate, if removal of type-1 and type-2 CALR mutations can ameliorate MPNs, we utilized our KI strategy to correct both CALR mutations in MPN patient-derived HSPCs by replacing them with wildtype sequences. A successful correction was confirmed on DNA and RNA level and by the absence of mutant CALR protein. Opposite to the results from introducing CALR mutations, correcting the mutations led to a two-fold decrease in megakaryocyte colony formation. Interestingly this was only seen in ET and post-ET MF samples, whereas primary MF samples were unaffected, underscoring the importance of other secondary genetic driver events in the pathogenesis of primary MF.Conclusion:Our system allows us to investigate human MPN pathogenesis prospectively and shed light on the transforming mechanisms of mutant CALR in primary HSPCs. We could show that CALR mutations prime HSPCs toward the formation of platelet-producing megakaryocytes. Genetic correction of CALR mutations in MPN patient-derived HSPCs revealed a dependence on the oncogenic mutant CALR driver event in ET and post-ET MF patients, opening the possibility of an ex vivo gene correction approach to remove mutant CALR in patient-derived HSPCs . Lastly, since MPN patient-derived cells have notoriously low engraftment potential in mice, our CRISPR/Cas9-engineered CALR mutant model also provides a powerful new strategy to generate MPN xenotransplants with defined genotypes for the evaluation of novel therapies. DisclosuresGreinix: Celgene: Consultancy; Therakos: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Novartis: Consultancy. Sill: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zebisch: Novartis: Consultancy; AbbVie: Consultancy; Celgene: Consultancy, Honoraria. Reinisch: Celgene: Research Funding; Pfizer: Consultancy.

A Calreticulin Neoepitope-Directed Monoclonal Antibody Can Overcome JAK Inhibitor Resistance and Block TPO-Independent Megakaryocyte Differentation

Introduction: Mutations within the gene encoding calreticulin (CALR) are the second most common genetic aberration associated with primary myelofibrosis (PMF), observed in 70% of non-JAK2 V617F cases. Importantly, patients with CALR mutations do not effectively respond to JAK inhibitor therapy and no mutation specific therapy is currently in use. Virtually all CALR mutations identified in PMF are small insertions or deletions clustered within exon 9 leading to a neo-epitope peptide sequence which is thought to directly or indirectly activate the thrombopoietin receptor (TpoR) by a poorly defined mechanism. Here we engineered a neo-epitope specific monoclonal antobody that has striking biological activity against ruxolitinib persistent cells.Methods TF-1 TpoR cells expressing TpoR were supplemented with 20 ng/mL of TPO. Rats were immunised with a CALR mutant peptide coupled to KLH. Serum from the immunised rats was screened by enzyme linked immunoassay, to verify a strong titre to the peptide immunogen. Primary PMF CD34+ cells were cultured in StemCell Pro with human SCF, IL-6 and IL-9. NSG mice were used to for engraftment studies after 150 cGy irradiation.Results: We engineered a panel of rat monoclonal antibodies after immunization with a 30 amino acid peptide corresponding to the C-terminal mutant CALR neoepitope sequence with an extra cysteine residue. Clone 4D7 showed superior activity of detecting mutant but not wild type CALR protein with a binding affinity of 13.5 pM and dissociation constant of 1.53 nM as measured by I 125-Scatchard. Treatment with 4D7 resulted in a significant (5-7-fold) increase in the amount of full-length mutant CALR protein in conditioned media. 4D7 inhibited Tpo-independent cell growth over 6 days in TF-1 cells expressing MPL and mutant CALR at 2, 10 and 20 µg. 4D7 blocked constitutive factor-independent phospho-STAT5 and phospho-ERK after incubation exclusively in mutant CALR cells but not in TF-1 cells expressing TpoR alone and increased the sub-G 0 fraction was observed compared to IgG control (P = 0.001, n = 3 independent experiments) consistent with induction of an apoptotic response. We tested activity in purified primary CD34+ cells obtained from patients with CALR mutant myelofibrosis using two orthogonal assays: - (i) Tpo-independent megakaryocyte differentiation in liquid culture and (ii) Tpo-independent megakaryocyte colony formation on a collagen-based medium. 4 out of 4 patient samples that displayed robust Tpo-independent growth of CD41+CD61+ megakaryocyte progenitors showed inhibition by 4D7 of at least 50%. Similarly, we saw dramatic reduction in the absolute numbers of primary Tpo-independent megakaryocyte colonies cultured on collagen (colony-forming unit-mega) treated with 4D7 in multiple patient samples (decrease of 46%, P = 0.0001, Student's t-test, n = 4 independent patient samples) Importantly, secretion of mutant CALR protein was neither upregulated nor downregulated by ruxolitinib, indicating ruxolitinib is unlikely to alter mutant CALR trafficking in patients. 4D7 had strong inhibitory activity on cells that were resistant to ruxolitinib, in both liquid culture at 96 hours or colony formation. To test whether 4D7 could block mutant CALR-dependent proliferation in vivo, we developed two distinct xenograft models, a bone marrow engraftment model, which measures mutant CALR dependent proliferation in the bone marrow microenvironment, and a chloroma model, which mimics extravascular infiltration of mutant CALR leukaemia, by injection of TPO-independent TF-1 cells in NSG mice. In the bone marrow engraftment model 4D7 treatment (12 mg/kg twice weekly via intraperitoneal injection) lowered peripheral blood engraftment of human CD33 myeloid cells at 3 weeks, bone marrow engraftment and significantly prolonged survival compared to IgG control (P=0.004, HR=0.2). In the chloroma model, 4D7 treatment resulted in significant decrease in tumour growth measured at 3 weeks (P<0.01) and improved overall survival (P=0.02, HR=0.07) compared to IgG controlConclusion: Together, these results suggest an immunotherapeutic approach may have clinical utility CALR-driven myeloproliferative neoplasms and CALR mutant acute myeloid leukaemia, as well as activity in CALR mutant patients that develop resistance/persistence to ruxolitinib. DisclosuresRoss: Bristol Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Keros Therapeutics: Consultancy, Honoraria. Reinisch: Celgene: Research Funding; Pfizer: Consultancy.

Pro-Inflammatory Cytokine IL-1b on Megakaryopoiesis: A Possible Explanation of Thrombocytosis in Inflammation

Pro-inflammatory cytokine interleukin-1b (IL-1b) is multifunctional and exerts its effects on a wide variety of cell types. Among many functions, IL-1b enhances the hematopoietic system by inducing T and B cell proliferation and works in combination with other cytokines on early stem and progenitor cell expansion and differentiation. Megakaryopoiesis and platelet production is maily controlled by thrombopoietin (TPO, Mpl-ligand), which is a growth factor for the megakaryocyte/ platelet lineage. Other early acting cytokines including IL-1, IL-3, IL-6 and IL-11 are also involved in the regulation of this lineage at different degree. Thrombocytosis is observed in various inflammation diseases. IL-1b, as a highly pro-inflammatory cytokine, induces thrombocytosis in mice. However, its mechanism on megakaryopoiesis is still not well understood. Our hypothesis is that IL-1b possesses a direct stimulatory effect on megakaryocytes via its receptors and the transcription factor NF-E2. The expression of IL-1 type I (IL-1 RI) and type II receptors was studied in megakaryotic cell lines: Meg-01, Dami, CHRF-288-11 and M-07e by RT-PCR, southern blot hybridization, flow cytometry or immuno-fluorescent staining. The effect of IL-1b on the formation of megakaryocytic colonies from human and murine stem and progenitor cells was investigated by the plasma clot culture method. The expression of the transcription factor p45 NF-E2, in response to IL-1b, was analyzed by Western blot. Our results show that IL-1 RI and IL-1 RII were expressed in the four human megakaryocytic cells. IL-1b alone or in combination with TPO induced megakaryocyte colony forming units (CFU-MK) in the culture system. The mitogenic effect of IL-1b on the enhancement of TPO-induced megakaryopoiesis was stronger than that of IL-3, IL-6, Flt-3 ligand and stem cell factor. The expression of NF-E2 was demonstrated in all four cell lines. In Meg-01 cells, the expression of NF-E2 was increased at both transcriptional and translational level after treatment with IL-1b for 4 hours. These observations strengthen the evidence of the direct effect of IL-1b on megakaryopoiesis. Our findings also provide a possible explanation of thrombocytosis during inflammation conditions. DisclosuresNo relevant conflicts of interest to declare.

Synthesis of Inflammatory Cytokines and Expression of Adhesion Molecules in Megakaryocytes

Inflammatory cytokines and platelet adhesion molecules may play a major role in hemostasis and immuno-pathological processes. Hematopoietic cells of myeloid and lymphoid lineages are known to be important sources of a variety of inflammatory cytokines. However, the immunological role of megakaryocytes is not well known. This project studied the involvement of megakaryocytes on immuno-inflammatory processes and the possible mechanism via the adhesion molecules and the synthesis of relevant cytokines. The expression of interleukin (IL)-1 to IL-10, TNF-a, TNF-g and IFN-g in primary megakaryocytes and four megakaryocytic cell lines: Meg-01, Dami, CHRF-288-11 and M-07e were determined by RT-PCR. The effect of IL-1b, IL-3, IL-6 and TPO on megakaryocyte colony formation (CFU-MK) was studied by using a plasma clot culture system. The CFU-MK was confirmed by AchE staining. The expression of adhesion protein CD36 on human platelets, megakaryocytes and megakaryocytic cell lines was also analyzed by using flow cytometry, ELISA and immunocytochemical methods. Our results showed that CD36 was expressed on platelets, megakaryocytes and the four megakaryocytic cell lines. The relative expression level is as follows: platelets > megakaryocytes > Meg-01 > Dami > CHRF-288-11 > M-07e, suggesting that the level of CD36 expression correlates with the degree of maturity of megakaryocytic differentiation. Inflammatory cytokines TNF-a, IL-1b, IL-3 and IL-6 were detected in primary megakaryocytes and all the four megakaryocytic cell lines, suggesting that different stages of megakaryocytes can be as a source of inflammatory cytokines. IL-1b, IL-3 and IL-6, as well as TPO, play a stimulating effect on CFU-MK formation, suggesting that there is an “autocrine” effect on megakaryopoiesis. Megakaryocytes may involve in immuno-inflammatory processes via the synthesis of inflammatory cytokines and platelet adhesion molecules. DisclosuresNo relevant conflicts of interest to declare.

The thrombopoietin mimetic JNJ-26366821 increases megakaryopoiesis without affecting malignant myeloid proliferation

Thrombocytopenia remains a challenge in myeloid malignancies, needing safer and more effective therapies. JNJ-26366821, a pegylated synthetic peptide thrombopoietin (TPO) mimetic not homologous to endogenous TPO, has an in-vitro EC50 of 0.2 ng/mL for the TPO receptor and dose dependently elevates platelets in volunteers. We demonstrate that JNJ-26366821 increases megakaryocytic differentiation and megakaryocytic colony formation in healthy controls and samples from myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). JNJ-26366821 had no effect on proliferation of malignant myeloid cell lines at doses up to 1000 ng/mL and malignant patient-derived mononuclear cells showed no increased cell growth or leukemic colony formation capacity at concentrations between 0.2 ng/mL and 10 ng/mL. Furthermore, JNJ-26366821 did not enhance in-vivo engraftment of leukemic cells in an AML xenotransplantation murine model. Our results show that JNJ-26366821 stimulates megakaryopoiesis without causing proliferation of the malignant myeloid clones in MDS/AML and provides the rationale for clinical testing of JNJ-26366821 in myeloid malignancies.

Suppression of Hematopoietic Primitive Cells in Patients with Secondary Failure of Platelet Recovery after Acute Graft-versus-Host Disease.

Secondary failure of platelet recovery (SFPR) can occur after allogeneic hematopoietic stem cell transplantation (alloHSCT), and 20% of cases are related to acute graft-versus-host disease (aGVHD). The underlying mechanisms of this association are unclear, however. The aim of the present study was to investigate the potential mechanisms of SFPR secondary to aGVHD, which may provide a new therapeutic strategy for these patients. A total of 468 patients with malignant hematologic disease who underwent alloHSCT were included. Sixty-six patients developed SFPR after alloHSCT, and in 45 of these 66 patients (68.2%), SFPR was secondary to grade II-IV aGVHD (SFPR/aGVHD). Compared with patients with good graft function (GGF), patients with SFPR had poor overall survival (20.72% versus 88.01%; P < .0001). Grade II-IV aGVHD was identified as an independent risk factor for SFPR in multivariate analysis (hazard ratio, 9.512; P < .0001). We observed reduced erythroid and megakaryocyte colony formation in bone marrow (BM) samples isolated from SFPR/aGVHD patients, consistent with the lower frequency of megakaryocyte and erythrocyte progenitors in BM. Levels of the inflammatory cytokines IL-2R and TNF-R1 were significantly higher in the SFPR/aGVHD group compared with the GGF group (P=.002 and .001, respectively), as were the frequencies of proinflammatory T helper subsets. Furthermore, the pathways that regulate hematopoiesis and immune responses were universally underexpressed in CD34+ cells isolated from SFPR/aGVHD patients. Differentially expressed genes were significantly enriched in the hematopoietic cell lineage pathway and other pathways involved in both immune responses and megakaryopoiesis. In summary, we found that both the immune microenvironment and compromised proliferation of hematopoietic primitive cells contribute to the development of SFPR secondary to aGVHD, and our data provide new insight into the mechanisms of SFPR in the context of aGVHD.

PF337 ALRN‐6924, A DUAL INHIBITOR OF MDMX AND MDM2 THAT CAUSES MINIMAL THROMBOCYTOPENIA IN PATIENTS, DISRUPTS DIFFERENT STAGES OF THROMBOPOIESIS COMPARED TO MDM2‐ONLY INHIBITION

Background:ALRN‐6924 is a cell‐permeating α‐helical peptide that disrupts interactions of the p53 tumor suppressor protein with its endogenous inhibitors, MDMX and MDM2. ALRN‐6924 can thereby restore p53‐dependent anti‐cancer activity in p53 wild type tumors. In an ongoing ALRN‐6924 clinical trial with 96 solid tumor and lymphoma patients enrolled as of 13Aug2018 there have been 1/0 adverse events (AE) of Grade 3/4 thrombocytopenia (TCP), respectively, at doses that yield objective responses (NCT02264613; Meric‐Bernstam et al, ASCO 2017; Shustov et al, ASH 2018). In contrast, small molecule MDM2‐only inhibitors induce Grade 3–4 TCP as a common AE and dose‐limiting toxicity.Aims:To explore the differences in TCP grades and frequencies from published solid tumor trials for ALRN‐6924 and MDM2i's, we investigated the effects of dual MDM2/MDMX‐inhibition vs. MDM2‐only inhibition during different stages of megakaryocyte maturation.Methods:Clonogenicity was measured in megakaryocyte (MK), erythroid (E), and granulocyte/monocyte myeloid (GM) progenitor colony‐forming unit (CFU) assays. Human bone marrow CD34+ cells seeded in methylcellulose semi‐solid media containing IL‐3 and IL‐6; and TPO, Epo or GM‐CSF together with ALRN‐6924, RG7388 (idasanutlin), or controls. CFU‐MK, CFU‐E, and CFU‐GM colonies were measured after 14 days of culture. Megakaryocyte maturation was modeled in vitro using a 14‐day liquid culture assay. Human bone marrow CD34+ cells were cultured in liquid medium containing SCF and TPO. Megakaryocyte maturation was assessed by measuring the expression of CD41 and CD42. To assess the effects of ALRN‐6924 and RG7388 on platelet development, drugs were added to the culture at different stages during the 14‐day period of megakaryocyte maturation in vitro. DNA synthesis was measured by BrdU, and apoptosis by Annexin V/propidium iodide. Healthy C57BL/6 mice treated with ALRN‐6924 3x/week were monitored for blood counts 2x/week; hematopoietic stem and progenitor cells in bone marrow were measured at 4 weeks.Results:In vitro, ALRN‐6924 disrupted MK colony formation more potently than RG7388, with IC50's of 0.024 and 0.12 μM, respectively, while their effects were comparable in CFU‐E and CFU‐GM assays with IC50's for both of ≈ 0.2 μM. Conversely, RG7388 disrupted megakaryopoiesis by inducing apoptosis and cell cycle arrest in maturing MKs 10‐fold more potently than ALRN‐6924. Consistent with these findings, ALRN‐6924 induced minimal effects on platelet counts in mice at doses that are effective in xenotransplanted and syngeneic tumor models, despite a significant decrease in MK and erythroid progenitors, while more immature hematopoietic stem and progenitor cells numbers remained unperturbed.Summary/Conclusion:ALRN‐6924 potently inhibits the clonogenic capacity of megakaryocyte progenitors in MK‐CFU assays but it is less potent during the process of megakaryocyte maturation than the MDM2‐only inhibitor RG7388. These findings suggest that the disruption of later stages of megakaryocyte maturation more severely affects platelet production, ultimately resulting in thrombocytopenia. However, inhibition of less mature megakaryocyte progenitors, prior to entering terminal maturation, appears to be better tolerated. This may explain the differences in TCP grades and frequencies from published solid tumor trials for ALRN‐6924 vs. MDM2‐only inhibitors, and suggests that MDMX in bone marrow cells, where MDMX RNA expression is the highest of all 37 normal tissues reported in the Protein Atlas, may contribute to these differences.

Modeling Calreticulin-Mutant Myeloproliferative Neoplasms with Isogenic Induced Pluripotent Stem Cells

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. DisclosuresIancu-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.

Protective Effect of the c-mpl Agonist Romiplostim on Megakaryocytopoiesis of Human CD34+Hematopoietic Progenitor Cells Exposed to Ionizing Radiation

A thrombopoiesis-stimulating protein, the myeloproliferative leukemia virus protooncogene (Mpl) ligand romiplostim (RP), is currently approved as a therapeutic agent for idiopathic thrombocytopenic purpura in many countries. Although the action of the initial MPL ligand thrombopoietin (TPO) on human megakaryocytic regeneration from irradiated human hematopoietic stem cells has been examined, there are few reports on the action of RP. In the present study, freshly prepared nonirradiated and 2-Gy X-irradiated human CD34 positive (CD34+) cells from placental umbilical cord blood were cultured with a combination of RP and various cytokines. As a result, the effect of RP on cell proliferation of nonirradiated CD34+ cells was found to be comparable to that of TPO. However, the stimulating activity of RP on megakaryocytic progenitor-derived colony formation was markedly lower compared with TPO. Regarding the action of RP with various cytokines, the present results showed that a combination of RP with interleukin-3 (IL-3) or IL-3 plus stem cell factor (SCF) showed a high regenerative effect on cell proliferation, megakaryopoiesis, thrombopoiesis, and megakaryocyte colony formation from X-irradiated CD34+ cells. The present study showed that human recombinant RP has potential effects on human megakaryocytic regeneration from X-irradiated human CD34+ cells and synergistically acts with IL-3 and IL-3 plus SCF, just as observed with TPO.

Development of a new knock-in mouse model and evaluation of pharmacological activities of lusutrombopag, a novel, nonpeptidyl small-molecule agonist of the human thrombopoietin receptor c-Mpl

Lusutrombopag (S-888711), an oral small-molecule thrombopoietin receptor (TPOR) agonist, has gained first approval as a drug to treat thrombocytopenia of chronic liver disease in patients undergoing elective invasive procedures in Japan. Preclinical studies were performed to evaluate its efficacy against megakaryopoiesis and thrombopoiesis. To investigate the proliferative activity and efficacy of megakaryocytic colony formation via human TPOR, lusutrombopag was applied to cultured human c-Mpl-expressing Ba/F3 (Ba/F3-hMpl) cells and human bone marrow-derived CD34-positive cells, respectively. Lusutrombopag caused a robust increase in Ba/F3-hMpl cells by activating pathways in a manner similar to that of thrombopoietin and induced colony-forming units-megakaryocyte and polyploid megakaryocytes in human CD34-positive cells. Because lusutrombopag has high species specificity for human TPOR, there was no suitable experimental animal model for drug evaluation, except for immunodeficient mouse-based xenograft models. Therefore, a novel genetically modified knock-in mouse, TPOR-Ki/Shi, was developed by replacing mouse Mpl with human-mouse chimera Mpl. In TPOR-Ki/Shi mice, lusutrombopag significantly increased circulating platelets in a dose-dependent manner during 21-day repeated oral administration. Histopathological study of the TPOR-Ki/Shi mice on day 22 also revealed a significant increase in megakaryocytes in the bone marrow. These results indicate that lusutrombopag acts on human TPOR to upregulate differentiation and proliferation of megakaryocytic cells, leading to platelet production.

Bone marrow-on-a-chip: Long-term culture of human haematopoietic stem cells in a three-dimensional microfluidic environment.

Multipotent haematopoietic stem and progenitor cells (HSPCs) are the source for all blood cell types. The bone marrow stem cell niche in which the HSPCs are maintained is known to be vital for their maintenance. Unfortunately, to date, no in vitro model exists that accurately mimics the aspects of the bone marrow niche and simultaneously allows the long-term culture of HSPCs. In this study, a novel three-dimensional coculture model is presented, based on a hydroxyapatite coated zirconium oxide scaffold, comprising of human mesenchymal stromal cells (MSCs) and cord blood derived HSPCs, enabling successful HSPC culture for a time span of 28days within the microfluidic multiorgan chip. The HSPCs were found to stay in their primitive state (CD34+ CD38- ) and capable of granulocyte, erythrocyte, macrophage, megakaryocyte colony formation. Furthermore, a microenvironment was formed bearing molecular and structural similarity to the in vivo bone marrow niche containing extracellular matrix and signalling molecules known to play an important role in HSPC homeostasis. Here, a novel human in vitro bone marrow model is presented for the first time, capable of long-term culture of primitive HSPCs in a microfluidic environment.

Signals of the Inflammatory Microenvironment Promote a Mutation-Associated Functional Dysregulation of the Circulating Megakaryocyte Progenitors of Myelofibrosis

Introduction: Myelofibrosis (MF), a clonal disorder of the hemopoietic stem/progenitor cell, is characterized by distinctive abnormalities in megakaryocyte (MK) development. A proliferation of dysplastic MK with disturbance of nuclear/cytoplasmic maturation is observed. In addition to driver mutations in Janus Kinase 2 (JAK2) and Calreticulin (CALR) genes, chronic inflammation has emerged as a key-player in MF pathogenesis. Here, we analyzed the potential contribution of crucial factors of the inflammatory microenvironment (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α and Tissue Inhibitor of Metalloproteinases (TIMP)-1 to MF dysmegakaryocytopoiesis.Methods: We studied 10 patients with MF at diagnosis while 6 patients had received previous treatment for MF. In all cases, therapies had been discontinued for at least two months before peripheral blood samples collection. To characterize the mutational status, JAK2V617F and CALR exon 9 mutations were assessed by quantitative PCR-based allelic discrimination assay and Next Generation Sequencing approach, respectively. To quantify MK progenitors, circulating CD34+CD41+ cells of MF patients (10 JAK2V617F and 6 CALR mutated) and controls (cord blood (CB); 8 cases) were identified by flow cytometry analysis. To evaluate the pro-inflammatory profile, IL-1β, TNF-α and TIMP-1 were measured in plasma of patients (10 JAK2V617F and 6 CALR mutated) and healthy subjects (10 cases) by ELISA. To determine whether and to what extent crucial factors of the inflammatory microenvironment may affect the in vitro growth of MK progenitors, Colony Forming Units-Megakaryocyte (CFU-MK) growth of circulating CD34+ cells from MF patients (6 JAK2V617F and 6 CALR mutated) and CB (6 cases) was assessed with a collagen-based medium in the presence or absence of TIMP-1, TNF-α and IL-1β (alone or in combination). Pure and mixed CFU-MK were quantified on the basis of CD41 immunostaining.Results: We found that, irrespective of mutational status, IL-1β, TNF-α and TIMP-1 plasma levels were significantly increased in MF patients. The frequency of circulating MF-derived CD34+CD41+ cells was significantly higher than the CB counterparts. However, a significantly higher proportion of cells was associated with CALR compared to JAK2V617F mutated patients.The growth of pure CFU-MK from untreated cells of the JAK2V617F mutated patients was significantly decreased as compared to that of CALR mutated patients and the CB counterparts. Factors alone did not stimulate the CFU-MK growth from CB CD34+ cells. Conversely, IL-1β had stimulatory activity on pure MK colony formation of JAK2V617F, but not CALR, mutated patients and the growth of MK progenitors from MF patients was significantly inhibited by TNF-α, irrespective of mutational status. TIMP-1 was ineffective. Factors in combination did not significantly modify the growth of pure CFU-MK from patients/CB as compared with factors alone. The growth of mixed MK colonies from untreated cells was absent (JAK2V617F mutated patients) or very low (CALR mutated patients and CB). Only TNF-α (alone or in combination (IL-1β+TNF-α and IL-1β+TNF-α+TIMP-1)) increases the growth of mixed MK colonies of patients harboring CALR mutation and CB. Interestingly, factors alone or in combination failed to stimulate the growth of mixed MK colonies from JAK2V617F mutated patients.Conclusions: Taken together these results demonstrate that in MF signals provided by the inflammatory microenvironment promote a mutation-associated functional dysregulation of MK progenitors and suggest a mechanism by which chronic inflammation may play a role in the development/maintenance of the abnormal megakaryocytopoiesis. DisclosuresMartinelli:BMS: Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Roche: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Genentech: Consultancy; Celgene: Consultancy, Speakers Bureau. Cavo:Janssen-Cilag: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria.

Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants

AbstractMutations in the calreticulin gene (CALR) represented by deletions and insertions in exon 9 inducing a −1/+2 frameshift are associated with a significant fraction of myeloproliferative neoplasms (MPNs). The mechanisms by which CALR mutants induce MPN are unknown. Here, we show by transcriptional, proliferation, biochemical, and primary cell assays that the pathogenic CALR mutants specifically activate the thrombopoietin receptor (TpoR/MPL). No activation is detected with a battery of type I and II cytokine receptors, except granulocyte colony-stimulating factor receptor, which supported only transient and weak activation. CALR mutants induce ligand-independent activation of JAK2/STAT/phosphatydylinositol-3′-kinase (PI3-K) and mitogen-activated protein (MAP) kinase pathways via TpoR, and autonomous growth in Ba/F3 cells. In these transformed cells, no synergy is observed between JAK2 and PI3-K inhibitors in inhibiting cytokine-independent proliferation, thus showing a major difference from JAK2V617F cells where such synergy is strong. TpoR activation was dependent on its extracellular domain and its N-glycosylation, especially at N117. The glycan binding site and the novel C-terminal tail of the mutant CALR proteins were required for TpoR activation. A soluble form of TpoR was able to prevent activation of full-length TpoR provided that it was N-glycosylated. By confocal microscopy and subcellular fractionation, CALR mutants exhibit different intracellular localization from that of wild-type CALR. Finally, knocking down either MPL/TpoR or JAK2 in megakaryocytic progenitors from patients carrying CALR mutations inhibited cytokine-independent megakaryocytic colony formation. Taken together, our study provides a novel signaling paradigm, whereby a mutated chaperone constitutively activates cytokine receptor signaling.

Adipose-Derived Mesenchymal Stem Cells with Potential to Ameliorate Platelet Recovery and Enhance Megakaryopoiesis in a Mouse Model of Radiation-Induced Thrombocytopenia Via the PI3k/Akt Pathway

▪*XHZ and JMZ contribute equally to this work# Co-correspondence to: Xiao-hui Zhang and Xiao-Jun HuangE-mail: zhangxh100@sina.comIntroduction: Substantial damage to the bone marrow can be caused by exposure to radiation, which can then develop into pancytopenia, especially severe thrombocytopenia. Severe thrombocytopenia can be a major, life-threatening event in untreated individuals. Recent studies have highlighted the role that radiation-induced thrombocytopenia plays in radiation mortality. However, studies focused on mitigating radiation-induced thrombocytopenia have rarely been reported. Among all alternative therapies available to patients experiencing radiation-induced thrombocytopenia, platelet transfusion is most effective. As a supportive therapy, platelet transfusion cannot substantially reconstitute the damaged hematopoietic properties of the bone marrow. Adipose-derived mesenchymal stem cells (ADSCs) are capable of migrating to injured tissue sites for damage repair. Moreover, it was demonstrated that ADSCs could support hematopoiesis both in vitro and in vivo. However, the therapeutic effects of ADSCs in radiation-induced thrombocytopenia as well as the underlying mechanism remain unknown. In this study, we hypothesized that administration of ADSCs may mitigate thrombocytopenia after radiation exposure. We investigated the in vivo impact of ADSCs on megakaryopoiesis and platelet recovery, and whether this amelioration effect was mediated through the activation of PI3K/Akt pathway in irradiated mice.Methods: The mouse model of radiation-induced thrombocytopenia was established by first irradiating mice with a 4Gy dose. Then, 15 mice were immediately injected with suspended ADSCs (1×106cells in 0.3 ml) and another 15 mice with equivalent saline. Ten unirradiated mice served as a control group. Platelet counts and white blood cell (WBC) counts in the peripheral blood were detected every week. Total colony formation units (CFU), megakaryocyte colony formation units (MK-CFU) and CD41+ cells in the bone marrow were assessed 21 days after irradiation. Bone marrow cellularity was determined by hematoxylin and eosin (HE) staining, and apoptosis was detected by terminal-deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) assay. Western blots were performed with anti-phosphorylated Akt, anti-Bcl-2 and anti-bax antibodies.Results: ADSCs obtained from the inguinal adipose exhibited a fibroblast-like morphology. Cultured cells were positive for CD29 (99.79%) and CD90 (97.82%), but not CD34 (3.08%), CD45 (1.04%) and CD31 (3.08%).Radiation markedly reduced peripheral blood counts, with a nadir on day 7. Recovery of both platelets (546.33±62.99 vs 375.48±50.33×109/L, P<0.05) and WBCs (4.23±0.51 vs 2.46±3.10×109/L, P<0.05) were better in the ADSCs-treated group compared with the saline group, respectively, 21 days after irradiation. A significant increase in total CFU (34.55±4.21 vs 12.86±2.15, P<0.05) and MK-CFU (6.28±0.74 vs 2.54±0.39, P<0.05) after irradiation were observed in the ADSCs group compared with the saline group, respectively. Further, the proportion of CD41+ cells in the ADSCs group was significantly higher than that in the saline group (4.2%±0.54% vs 1.21%±0.11%, P<0.05).A radioprotective effect was shown in the ADSCs-treated group, especially in the megakaryocytic lineage, by HE staining. In the ADSCs group, the number of apoptotic cells was significantly lower than that in the saline group (3.52±0.42 vs 13.48±2.15 per field, P<0.05). Administration of ADSCs up-regulated protein expression of phosphorylated Akt and Bcl-2, whereas the expression of Bax, a protein related to apoptosis, was significantly lower in the ADSCs group.Conclusion:For the first time, we demonstrated that ADSCs were capable of promoting platelet recovery and improving megakaryopoiesis in irradiated mice. This protective function of ADSCs is likely to be mediated via the PI3K/Akt pathway, which would thus provide a new therapeutic alternative for mitigating radiation-induced thrombocytopenia. DisclosuresNo relevant conflicts of interest to declare.

MEIS1 regulates early erythroid and megakaryocytic cell fate.

MEIS1 is a transcription factor expressed in hematopoietic stem and progenitor cells and in mature megakaryocytes. This biphasic expression of MEIS1 suggests that the function of MEIS1 in stem cells is distinct from its function in lineage committed cells. Mouse models show that Meis1 is required for renewal of stem cells, but the function of MEIS1 in human hematopoietic progenitor cells has not been investigated. We show that two MEIS1 splice variants are expressed in hematopoietic progenitor cells. Constitutive expression of both variants directed human hematopoietic progenitors towards a megakaryocyte-erythrocyte progenitor fate. Ectopic expression of either MEIS1 splice variant in common myeloid progenitor cells, and even in granulocyte-monocyte progenitors, resulted in increased erythroid differentiation at the expense of granulocyte and macrophage differentiation. Conversely, silencing MEIS1 expression in progenitor cells induced a block in erythroid expansion and decreased megakaryocytic colony formation capacity. Gene expression profiling revealed that both MEIS1 splice variants induce a transcriptional program enriched for erythroid and megakaryocytic genes. Our results indicate that MEIS1 expression induces lineage commitment towards a megakaryocyte-erythroid progenitor cell fate in common myeloid progenitor cells through activation of genes that define a megakaryocyte-erythroid-specific gene expression program.

Loss of Stat1 decreases megakaryopoiesis and favors erythropoiesis in a JAK2-V617F–driven mouse model of MPNs

AbstractThe interferon-γ (IFNγ)/signal transducer and activator of transcription 1 (Stat1) pathway shows higher activity in patients with essential thrombocythemia (ET) than in polycythemia vera (PV) and was proposed to be promoting the ET phenotype. We explored the phenotypic consequences of Stat1 deficiency on the effects of Janus kinase 2 (JAK2)–V617F in vivo by crossing mice expressing JAK2-V617F with Stat1 knockout mice. JAK2-V617F;Stat1−/− double transgenic mice showed higher red cell parameters and lower platelet counts compared with JAK2-V617F;Stat1+/+ mice. Bone marrow transplantation reproduced these phenotypic changes in wild-type recipients, demonstrating that the effect of Stat1 is cell-intrinsic and does not require a Stat1-deficient microenvironment. Deletion of Stat1 increased burst-forming unit–erythroid and reduced colony-forming unit–megakaryocyte colony formation driven by JAK2-V617F, but was not sufficient to completely normalize the platelet count. Gata1, a key regulator of megakaryopoiesis and erythropoiesis, was decreased in Stat1-deficient platelets. V617F transgenic mice with thrombocytosis had higher serum levels of IFNγ than normal controls and patients with ET showed higher IFNγ serum levels than patients with PV. Together, these results support the concept that activating Stat1 in the presence of JAK2-V617F, for example, through IFNγ, constrains erythroid differentiation and promotes megakaryocytic development, resulting in ET phenotype.

Heterozygous and Homozygous JAK2V617F States Modeled by Induced Pluripotent Stem Cells from Myeloproliferative Neoplasm Patients

JAK2V617F is the predominant mutation in myeloproliferative neoplasms (MPN). Modeling MPN in a human context might be helpful for the screening of molecules targeting JAK2 and its intracellular signaling. We describe here the derivation of induced pluripotent stem (iPS) cell lines from 2 polycythemia vera patients carrying a heterozygous and a homozygous mutated JAK2V617F, respectively. In the patient with homozygous JAK2V617F, additional ASXL1 mutation and chromosome 20 allowed partial delineation of the clonal architecture and assignation of the cellular origin of the derived iPS cell lines. The marked difference in the response to erythropoietin (EPO) between homozygous and heterozygous cell lines correlated with the constitutive activation level of signaling pathways. Strikingly, heterozygous iPS cells showed thrombopoietin (TPO)-independent formation of megakaryocytic colonies, but not EPO-independent erythroid colony formation. JAK2, PI3K and HSP90 inhibitors were able to block spontaneous and EPO-induced growth of erythroid colonies from GPA+CD41+ cells derived from iPS cells. Altogether, this study brings the proof of concept that iPS can be used for studying MPN pathogenesis, clonal architecture, and drug efficacy.