Abstract IDH1 and IDH2 are frequently mutated in various cancers, including acute leukemias. However, the distinct mechanisms by which mutant IDH1 or IDH2 drive hematopoietic neoplasms remain poorly understood. Here, we analyzed DNA methylation in IDH1- and IDH2-mutant acute myeloid leukemia and found neutrophil lineage-specific epigenetic alterations in IDH1-mutant patients that went along with severely impaired neutrophil differentiation. Transcriptional analysis of normal hematopoiesis in humans and mice revealed a strong physiological upregulation of IDH1/Idh1 in myeloid progenitors. To study the functional effects of Idh1 mutations on hematopoiesis in a preleukemic setting, we used a genetically engineered inducible mouse model expressing a heterozygous Idh1 mutation under control of the endogenous promotor. Our study revealed a cell-intrinsic block in neutrophil differentiation caused by repression of myeloid transcription programs in neutrophil progenitors. This included impaired expression of Cebpe, which encodes a key transcription factor regulating neutrophil differentiation. Reactivation of Cebpe expression, by overexpression of its upstream regulator Cebpa or following treatment with hypomethylating agents, restored differentiation, indicating that the differentiation block is reversible. In summary, we found a reversible, preleukemic impairment of neutrophil differentiation in IDH1-mutant hematopoiesis that correlates with elevated IDH1 expression in myeloid progenitors and likely explains the strong association of IDH1 mutations with myeloid neoplasms.

Persistent fetal gene expression in childhood neoplasms is usually explained by a maturation block originating in the prenatal phase. In contrast, reactivation of fetal genes in adult malignancies is considered a consequence of oncofetal reprogramming (OFR) and is associated with aggressive disease. By reconstructing epigenetic ontogeny in juvenile myelomonocytic leukemia (JMML), we identified a postnatal maturation state of JMML stem cells with high transcriptional plasticity indicative of OFR in high-risk disease. Similarly, postnatal activation of oncogenic signaling by inducible Ptpn11E76K mutation in mice triggered molecular plasticity and reactivation of fetal gene expression. Integrative multi-omics analysis revealed aberrant CD52 expression as a feature of high-risk JMML stem cells. Anti-CD52 treatment depleted JMML stem cells and blocked disease propagation in xenograft models. Our results challenge the prevailing maturation block model of pediatric leukemogenesis and establish RAS-associated stem cell plasticity as a determinant of OFR and potential therapeutic vulnerabilities in high-risk JMML. SIGNIFICANCE:Persistent fetal gene expression in pediatric malignancies is considered a consequence of prenatal maturation blockade. In this study, we demonstrate that oncogenic PTPN11 mutations enhance cellular plasticity. This leads to partial restoration of fetal molecular programs, creating new therapeutically exploitable vulnerabilities. See related commentary by Miao and Xu, pp. 168.

Platelet-biased hematopoietic stem cells (PLT-HSCs) play key roles in normal physiology, aging, and blood cancer. However, currently, no markers allow their accurate identification or prospective isolation. We here combine single-mouse hematopoietic stem cell (HSC) gene expression, chromatin accessibility, and surface proteome profiling to identify subtype-specific markers. Using machine learning, we identified markers (CD61hiCD274hiCD357loCD27lo) that isolate PLT-HSCs to high purity, validated by single-cell transplantation. Furthermore, we develop a minimal expression marker panel that discriminates PLT- and multi-lineage (MUL-)HSCs using microfluidics-based single-cell RT-qPCR. We show that both methods detect the age-associated increase in PLT-HSCs, while poly(I-C)-induced chronic inflammation did not alter HSC lineage bias. In contrast, romiplostim treatment increased MUL-HSC prevalence. Finally, using spectral flow cytometry to simultaneously quantify cell cycle and HSC lineage bias, we show that platelet depletion selectively activates PLT-HSCs. Together, these approaches allow accurate isolation of PLT-HSCs and robust quantification of lineage bias under perturbation.

Supplementary Data 1-5 from Reciprocal Relationship between O6-Methylguanine-DNA Methyltransferase P140K Expression Level and Chemoprotection of Hematopoietic Stem Cells

BACKGROUND & AIMS:BRAF-mutant colorectal cancer (CRC) is a clinically aggressive subtype arising from the serrated pathway and is associated with poor prognosis and therapy resistance. The mechanisms driving malignant transformation in microsatellite-stable (MSS) BRAF-mutant CRC remain incompletely understood. We aimed to define the role of WNT pathway activation in serrated CRC progression and tumor-immune interactions. METHODS:We generated multiple genetically engineered mouse models of BRAF-mutant MSS CRC and complementary organoid-based transplantation models. Genetic alterations in WNT pathway components were functionally interrogated. Tumor development and immune microenvironment remodeling were analyzed using bulk RNA sequencing, single-cell RNA sequencing, Cellular Indexing of Transcriptomes and Epitopes by Sequencing, and functional in vivo assays. RESULTS:WNT pathway activation via APC or CTNNB1 mutations, but not RNF43 loss, was required for tumor initiation in BRAF-mutant CRC models. WNT activation induced a molecular subtype shift and suppressed immune response pathways. Mechanistically, WNT signaling suppressed C-C motif chemokine ligand 20 expression and remodeled the tumor microenvironment by promoting immunosuppressive myeloid populations and altering T-cell states. Functional assays demonstrated that WNT activation enhances tumor progression in immunocompetent settings, indicating immune evasion as a key driver of malignant progression. CONCLUSIONS:WNT pathway activation is a critical determinant of malignant transformation in BRAF-mutant MSS CRC by enabling immune escape. These findings identify WNT signaling as a central regulator of tumor-immune interactions and a potential therapeutic target in this aggressive CRC subtype.

Abstract Genome instability and mutagenesis are hallmarks of aging, acting as drivers of some age-associated pathologies, including cancer 1–3 . Somatic cells engage multiple layers of protection against mutagenesis, including detoxification of genotoxic metabolites; repair of DNA damage; and elimination of cells which suffer excessive damage 4–7 . In this context, the intrinsic apoptotic pathway is engaged in response to activation of the DNA damage response (DDR) and is thought to play a major role in limiting accumulation of mutations, particularly in cells that act as an origin for cancer, such as somatic stem cells 8,9 . However, the dissection of the relative contribution of different protective mechanisms that restrict mutagenesis in such cells is confounded by the long time frame of experiments; relatively low mutation burden in non-malignant cells; and high variance across individuals due to differences in germ line and environment. Here we employ deep whole-genome sequencing (WGS) combined with extended time-course sampling from a range of experimental mouse models to study mutation acquisition in hematopoietic stem cells (HSCs) during aging. Having validated that murine HSCs recapitulate mutation acquisition patterns observed in aged human HSCs, we made the surprising discovery that apoptosis has a negligible role in restricting mutagenesis. Instead, we found that HSC dormancy inhibits mutagenesis during normal aging, with dormant HSCs from old mice demonstrating a mutation burden akin to their young counterparts. Importantly, breaking HSC dormancy via induction of sterile inflammation led to a dramatic acceleration in mutation rate, demonstrating that non-genotoxic environmental stimuli can modulate genome stability. These findings provide new insights into the correlation between inflammation and both aging and carcinogenesis.

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Haematopoietic stem cells (HSCs) display extensive molecular and functional heterogeneity. However, a cohesive model that explains the relationship and biological relevance of these diverse HSC states remains elusive. Here, by performing single-cell transplantations of over 1,000 highly purified murine long-term HSCs combined with in-depth phenotyping of their clonal progeny, we define kinetics-based reconstitution parameters which aligned HSCs into a single hierarchical trajectory reflective of functional potency. This approach revealed that previously identified lineage biases are actually transitory states along this linear trajectory, not a discrete stable condition. Single-cell secondary transplantations validated hierarchical ordering based on reconstitution kinetics, whereas mathematical modelling combined with experimental modulation of lineage-biased blood production revealed that apparent lineage-biased outputs actually arise from cell-extrinsic feedback regulation and clonal competition between slow- and fast-engrafting clones to fill mature lineages to their compartment size limit. This study reconciles multiple layers of HSC heterogeneity into a unifying framework.

Residing at the top of the hematopoietic hierarchy, long-term hematopoietic stem cells (HSCs) are capable of self-renewal and sustained blood cell regeneration. Over the past decades, single-cell and clonal analyses have revealed substantial functional and molecular heterogeneity within this compartment, challenging the notion that self-renewal is inherently tied to balanced, multi-lineage blood production. However, a cohesive model that explains the relationships among these diverse HSC states remains elusive. Here, we combined single-cell transplantations of over 1,000 highly purified murine long-term HSCs with in-depth phenotyping of their clonal progeny to achieve a detailed, time-resolved understanding of heterogeneous reconstitution outcomes. We identified reconstitution kinetics as an overall unifying metric of HSC functional potency, with the most potent HSCs displaying the greatest delay in hematopoietic reconstitution. Importantly, a progressive acceleration in reconstitution kinetics was also associated with a gradual shift in mature cell production from platelet and erythro-myeloid bias to balanced, and eventually lymphoid bias. Serial single-cell transplantations of HSCs revealed a unidirectional acceleration in reconstitution kinetics accompanied by a gradual decline in functional potency of daughter HSCs, aligning diverse phenotypes along a linear hierarchical trajectory. Mathematical modeling, together with experimental modulation of lineage-biased blood production, demonstrated that apparent lineage biases actually arise from cell-extrinsic feedback regulation and clonal competition between slow- and fast-engrafting clones to occupy the limited compartment sizes of mature lineages. Our study reconciles multiple layers of HSC heterogeneity into a unifying framework, prompting a reevaluation of the meaning of lineage biases in both normal and diseased hematopoiesis, with broad implications for other regenerating tissues during development, homeostasis, and repair. ### Competing Interest Statement The authors have declared no competing interest.

Long-term dormancy is proposed to mediate therapy resistance of cancer stem cells. However, the biological relevance of dormancy in hematologic malignancies has not been formally addressed. Using a novel mouse model that allows identification and isolation of dormant label retaining cells (LRCs) in the setting of JAK2V617F-driven Polycythemia Vera (PV), we could identify LRCs that maintained dormancy for at least 20 weeks in vivo in PV mice, despite harboring the pro-proliferative JAK2 mutation. Isolation and transplantation of mutant LRCs and nonLRCs unambiguously demonstrated that only LRCs were capable of propagating the PV phenotype. Treatment of primary PV mice with a JAK2 inhibitor (Fedratinib, FED) failed to target mutant LRCs, while pegylated interferon-a (IFNa) reduced their number, in line with the clinical observation that IFNa can facilitate molecular remission while FED cannot. After therapy, purified residual LRCs were still uniquely able to propagate PV, demonstrating their role as disease-reinitiating cells and that their level of depletion is a biomarker of PV eradication. Side effects of IFNa treatment limit its clinical effectiveness in PV. To circumvent this, we explored using low dose IFNa (IFNaLD) alone or in concert with FED. IFNaLD alone had no impact on mutant LRC numbers, while IFNaLD plus FED resulted in a synergistic depletion of mutant LRCS and correction of PV phenotype, suggesting that IFNaLD renders LRCs dependent upon JAK2 signaling. In summary, we provide definitive evidence of the importance of dormancy in propagation and therapy resistance of PV; create a model for pre-clinical evaluation of novel therapies for PV; and demonstrate that a combination of IFNaLD and FED may be effective at eliminating mutant LRCs while limiting side effects of standard IFNa treatment.

Mutations of Isocitrate dehydrogenase 1 (IDH1) are found in several malignancies, including myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML). Despite rarely being detected in clonal hematopoiesis, IDH1 mutations are considered to be early pathogenetic events. IDH1 mutations are always heterozygous and result in neomorphic enzymatic activity leading to the production of the oncometabolite D-2-hydroxyglutarate (D2HG). D2HG inhibits α-ketoglutarate-dependent dioxygenases resulting in DNA and histone hypermethylation. However, to date the intricate molecular alterations resulting from IDH1 mutations and how they contribute to malignant transformation are only poorly understood.The aim of this study was to investigate the cellular and molecular aberrations elicited by mutant Idh1 in hematopoiesis to better understand the mechanisms driving IDH1-mutant (mut) hematopoietic neoplasms.We established a conditional Idh1-R132H mouse model in which Idh1-R132H and YFP-reporter expression are induced in hematopoietic stem and progenitor cells (HSPC) using a tamoxifen-inducible Cre driven by the Scl enhancer (Scl-CreERT). Lineage-/YFP+ cells were transplanted into lethally irradiated mice. Blood and bone marrow cells from fully chimeric Idh1-wildtype (WT) and Idh1-R132H mice were subjected to scRNA-seq, and ex vivo/in vivo assays. The Idh1-R132H mutation was also expressed in 32D cells using lentiviral transduction. Publicly available DNA methylation data from AML patients were collected from TCGA (LAML), BEAT-AML and from Schmutz et al. (Clin Epigenetics 2023). Peripheral blood counts from MDS and AML patients were obtained from patient registries. Analysis of scRNA-seq data of normal human and murine bone marrow revealed dynamic regulation of IDH1/Idh1 expression in hematopoiesis with peak expression in myeloid progenitor (MP) cells. In contrast, expression of IDH2/Idh2 was observed uniformly across all hematopoietic stem and progenitor cell types. In Idh1-R132H mice, this upregulation strongly correlated with a significant expansion of MPs (CD55- CMPs and Ly6C+ GMPs) and a loss of neutrophils. Adoptive transfer confirmed that Idh1-R132H CD55- CMPs produce fewer neutrophils than their WT counterparts, suggesting that Idh1-R132H MPs exhibit a cell-intrinsic defect in granulopoiesis. This differentiation defect was reproduced in 32D cells expressing Idh1-R132H. Treatment of Idh1-R132H CD55- CMPs with a mutant-specific IDH1-inhibitor virtually reversed the differentiation defect in an ex vivo assay. Further, scRNA-seq revealed a depletion of late neutrophil progenitors in Idh1-R132H mice and reduced expression of the granulocyte transcription factor Cebpe, which was again validated in Idh1-mut 32D cells. Therefore, 32D cells were modified with a tamoxifen-inducible Cebpe constract (Theilgaard-Mönch et al., 2022). Tamoxifen treatment and with that translocation of CEBPE to the nucleus was sufficient to overcome the differentiation block of Idh1-mut cells. To test the relevance of our findings for AML patients, we compared DNA methylation data from IDH1-mut AML samples with data from normal human hematopoietic cell types. This revealed epigenetic scars of granulocytic lineage commitment in 9/16 IDH1-mut AML samples which were absent in IDH2-mut AML samples. This suggested that a granulocytic differentiation defect might contribute to leukemogenesis in IDH1-mut AML. In line with this we found that MDS and AML patients with IDH1-mut presented significantly lower neutrophil counts as compared to IDH2-mut and IDH1/2-WT patients. In contrast, no other cell lineage presented a significant change between IDH1- and IDH2-mut patients. In the present study, we identified cellular defects in MPs of Idh1-mut mice, which are mediated by a physiologic upregulation of Idh1 expression in MPs. This results in downregulation of Cebpe in neutrophil progenitors and impaired granulopoiesis. Re-introducing CEBPE activity is sufficient to overcome the differentiation block. Identification of granulocytic epigenetic scars in IDH1-mut AML samples and neutropenia in IDH1-mut MDS and AML patients suggest that neutrophilic differentiation defects might play a role in leukemogenesis in humans. In summary, our molecular and cellular data provide an explanation for the preferential association of IDH1 mutations with myeloid neoplasms.

Organismal aging is thought to be mediated by the interaction of multiple genetic and environmental variables acting cumulatively over long periods of time, confounding mechanistic insights into this process. In this study, we measured a wide range of hematologic variables (cell counts, histology, flow cytometry, HSC transplantation, scRNA and scATAC-seq) from hematopoietic tissues across a large cohort (>100 individuals) of young (8 weeks), middle aged (18 months) and old (>24 months) female C57BL/6J mice housed in the same controlled environment. Surprisingly, the aged phenotypes across the cohort were highly variable, with some 24-month-old mice displaying parameters in line with 8-week-old controls while others demonstrating extreme aged outcomes, despite the minimal variance in genotype and environment. This suggests a dominant stochastic basis to hematopoietic aging that is rarely considered in the literature. Importantly, canonical age-associated phenotypes that are thought to have a causal relationship (HSC functional potential, HSC expansion, myeloid bias, anemia) poorly correlated across the cohort, challenging the concept that HSC dysfunction drives the evolution of aged hematopoiesis. scRNAseq of bone marrow HSCs, progenitors, mature hematopoietic and niche cells identified a new population of inflammatory adipocytes precursors exclusive to, but heterogenous across aged individuals. Interaction analysis suggests that these cells receive inflammatory signals from neutrophils (Il1b and Tnf), and downregulate ligands (Kitl, Vcam1, and Angpt2) that typically signal to HSCs, potentially mediating HSC decline during aging. Taken together, these findings challenge the notion of a uniform hematological aging process stemming from compromised HSCs, but rather indicate a stochastic process, which extends to a heterogenous niche composition.

The earliest step of adult hematopoiesis involves the transition from hematopoietic stem cells (HSCs) to multipotent progenitors (MPPs). MPPs are highly heterogeneous and the hierarchical relationship between HSCs and individual MPP populations remains unsolved. In particular, little is known about the newly described MPP6s, which can be phenotypically defined as CD34–CD135–CD48–CD150–LSKs.Here, we discovered that EPCR expression discriminated EPCR– ‘MPP6s’ that closely resemble erythroid progenitors, from bona fide EPCR+ MPP6s that display extensive stem cell programs. MPP6s had superior serial repopulating capacity compared to MPP1-5s, with only a slight decrease in long-term (LT) myeloid output compared to HSCs in secondary transplantation. MPP6s were capable of self-renewing and reconstitution of all phenotypic HSC/MPP populations in the bone marrow 16 weeks post-transplantation. Furthermore, similar engraftment frequency was found in MPP6s and HSCs when transplanting at the single cell (sc) level.Interestingly, using an Scl-tTA; H2B-GFP mouse model, we found that the proportion of H2B-GFP+ LT label-retaining cells was consistently higher in MPP6s than in HSCs after 100 days of chase, demonstrating that MPP6s are highly dormant. To further evaluate the complex interplay between dormancy and developmental transition within the HSC/MPP network, we isolated GFP+ and GFP– HSC/MPPs and performed transplantation and sc-multiomics analysis using two platforms (scMT-seq and 10x multiome RNA+ATAC). Preliminary analysis at the RNA level revealed unbiased separation of dormant GFP+ and active GFP– cells, followed by secondary clustering of phenotypic HSC/MPP subsets. Collectively, our data suggest that there is plasticity on CD150 expression in homeostatic LT HSCs and suggests a larger functional HSC pool as so far anticipated that includes dormant EPCR+ MPP6 cells.

Acute myeloid leukemia (AML) driven by the activation of EVI1 due to chromosome 3q26/ MECOM rearrangements is incurable. Because transcription factors such as EVI1 are notoriously hard to target, insight into the mechanism by which EVI1 drives myeloid transformation could provide alternative avenues for therapy. Applying protein folding predictions combined with proteomics technologies, we demonstrate that interaction of EVI1 with CTBP1 and CTBP2 via a single PLDLS motif is indispensable for leukemic transformation. A 4× PLDLS repeat construct outcompetes binding of EVI1 to CTBP1 and CTBP2 and inhibits proliferation of 3q26/ MECOM rearranged AML in vitro and in xenotransplant models. This proof-of-concept study opens the possibility to target one of the most incurable forms of AML with specific EVI1-CTBP inhibitors. This has important implications for other tumor types with aberrant expression of EVI1 and for cancers transformed by different CTBP-dependent oncogenic transcription factors.

Aging exerts a profound impact on the hematopoietic system, leading to increased susceptibility to infections, autoimmune diseases, chronic inflammation, anemia, thrombotic events, and hematologic malignancies. Within the field of experimental hematology, the functional decline of hematopoietic stem cells (HSCs) is often regarded as a primary driver of age-related hematologic conditions. However, aging is clearly a complex multifaceted process involving not only HSCs but also mature blood cells and their interactions with other tissues. This review reappraises an HSC-centric view of hematopoietic aging by exploring how the entire hematopoietic hierarchy, from stem cells to mature cells, contributes to age-related disorders. It highlights the decline of both innate and adaptive immunity, leading to increased susceptibility to infections and cancer, and the rise of autoimmunity as peripheral immune cells undergo aging-induced changes. It explores the concept of "inflammaging," where persistent, low-grade inflammation driven by old immune cells creates a cycle of tissue damage and disease. Additionally, this review delves into the roles of inflammation and homeostatic regulation in age-related conditions such as thrombotic events and anemia, arguing that these issues arise from broader dysfunctions rather than stemming from HSC functional attrition alone. In summary, this review highlights the importance of taking a holistic approach to studying hematopoietic aging and its related pathologies. By looking beyond just stem cells and considering the full spectrum of age-associated changes, one can better capture the complexity of aging and attempt to develop preventative or rejuvenative strategies that better target multiple facets of this process.

The transient cellular state of dormancy is proposed to mediate cancer stem cell therapy resistance and subsequent disease relapse. However, dormancy is challenging to interrogate experimentally, and has not been formally demonstrated to exist in hematological malignancies. We have developed a novel transgenic mouse model that combines: (i) inducible expression of the JAK2-V617F mutation (JAK2VF), which drives a Polycythemia Vera-like (PV) disease; and (ii) an inducible H2B-GFP fusion protein, which facilitates prospective identification and isolation of live dormant JAK2VF hematopoietic stem cells (dHSCs) based on their long-term retention of H2BGFP-labelled chromatin once the expression of the fusion protein is switched off. To interrogate if dHSCs exist in vivo, we performed label retention experiments after activation of the JAK2VF mutation. During the chase period, all mice developed PV, demonstrating disease progression (hematocrit (HCT) >75%; splenomegaly with spleen weight >0.5 g). Despite the presence of the pro-proliferative JAK2VF mutation, almost a quarter of mutant HSCs maintained a dormant state for 12 weeks in vivo (22.4±3.7%), and dHSCs could still be identified after 20 weeks of chase (1.9±0.5%). To assess if dormancy held any biological relevance in cancer stem cells, we prospectively isolated both dHSCs and label-diluted “active” HSCs (aHSCs) from the same mice, and interrogated disease-initiating capacity following secondary transplantation of 150 dHSCs or aHSCs into each recipient mouse. While none of the mice transplanted with aHSCs developed disease, 90% of mice receiving dHSCs developed a PV phenotype within 8 weeks post-transplantation. We next employed our model to assess the relative impact of clinically relevant therapies on dHSCs by treating PV mice for 4 consecutive weeks with either the JAK2 inhibitor Fedratinib (FED) or Peg-IFN-α (IFN-α) during the label chase period. Both FED and IFN-α resolved splenomegaly (0.4±0.04g, 0.17±0.02g, 0.22±0.05g for control (CON), FED and IFN-α-treated, p < 0.0001 for CON vs. FED, p<0.0001 for CON vs. IFN-α) and partially corrected HCT levels (78±3.9%, 77±5%, 60±10.3% for CON, FED and IFN-α-treated, p<0.0001 for CON vs IFN-α). However, FED had no impact on the frequency of disease-propagating dHSCs in the bone marrow, while IFN-α significantly reduced their frequency (29.2±3%, 28.3±7%, 13.6±4% for CON, FED and IFN-α), suggesting that dHSCs may not be addicted to JAK2VF signaling for survival. This is consistent with the clinical observation that FED and IFN-α both resolve hematological parameters but have differential effects on JAK2VF allele burden. Importantly, residual dHSCs enriched by flow cytometry post-IFN-α therapy were still capable of propagating disease in secondary recipient mice, while aHSCs were not, indicating it may be necessary to completely eradicate dHSCs with prolonged therapy to achieve full remission. We next wished to employ this model as a pre-clinical tool to assess the effect of novel therapeutic regimens on dHSCs. We first examined whether halving the dose of IFN-α could still result in dHSC depletion (Low dose (LD)-IFN-α), based on the rationale that such an approach might circumvent side effects associated with IFN-α therapy leading to therapy discontinuation. However, LD-IFN-α only achieved a modest correction of hematological parameters while failing to deplete dHSCs. We next tested a novel combination therapy of LD-IFN-α plus FED. This combination resulted in a correction of HCT (78.9±2.6%, 61.5±4.1%, p=0.04 for CON vs. FED + LD-IFN-α) and splenomegaly (0.41±0.05 g, 0.19±0.02 g, p<0.001 for CON vs. FED + LD-IFN-α), as well as a reduction in dHSCs similar to the standard higher IFN-α dosage (29.2±3%, 15.6±4.5%, 13.6±3.4% for CON, FED+LD-IFN-α and IFN-α). This suggests that LD-IFN-α renders dHSCs reliant on JAK2 signaling and thus susceptible to JAK2 inhibition. In conclusion we have demonstrated the existence of a rare subpopulation of JAK2VF-mutant dHSCs that represents the PV disease propagating cell, which demonstrates differential susceptibility to JAK2 inhibition (resistant) and IFN-α (sensitive). As proof of concept of employing this model for pre-clinical evaluation of new therapeutic avenues, we have tested a novel combination therapy of FED plus LD-IFN-α which effectively depletes dHSCs while also correcting clinically-relevant hematological parameters.