Genetic barcoding systematically comparing genes in del(5q) MDS reveals a central role for CSNK1A1 in clonal expansion

How genetic haploinsufficiency contributes to the clonal dominance of hematopoietic stem cells (HSC) in del(5q) myelodysplastic syndrome (MDS) remains unresolved. Using a genetic barcoding strategy, a systematic comparison was carried out on genes implicated in the pathogenesis of del(5q) MDS in direct competition with each other and wild-type (WT) cells with single clone resolution. Csnk1a1 haploinsufficient HSCs expanded (oligo)clonally and outcompeted all other tested genes and combinations. Csnk1a1-/+ multipotent progenitors showed a pro-proliferative gene signature and HSCs a downregulation of inflammatory signaling/immune response. In validation experiments, Csnk1a1-/+ HSCs outperformed their WT counterparts under a chronic inflammation stimulus, also known to be caused by neighboring genes on chromosome 5. A crucial role for Csnk1a1 haploinsufficiency in the selective advantage of the 5q- HSC is therefore proposed. It is implemented by creation of a unique competitive advantage through increased HSC self-renewal and proliferation capacity, as well as increased fitness under inflammatory stress.

Rnf149-related is an FGF/MAPK-independent regulator of pharyngeal muscle fate specification

During embryonic development, cell fate specification gives rise to dedicated lineages that underlie tissue formation. In olfactores, which comprise tunicates and vertebrates, the cardiopharyngeal field is formed by multipotent progenitors to both cardiac and branchiomeric muscles. The ascidian Ciona is a powerful model to study the cardiopharyngeal fate specification with cellular resolution, as only 2 pairs of cardiopharyngeal multipotent progenitors give rise to the heart and to pharyngeal muscles (aka atrial siphon muscles, ASM). These progenitors are multilineage primed, in as much as they express a combination of early ASM- and heart-specific transcripts that become restricted to their corresponding precursors, following oriented asymmetric divisions. Here, we identify the primed gene Rnf149-related (Rnf149-r), which becomes restricted to the heart progenitors, but appears to regulate pharyngeal muscle fate specification in the cardiopharyngeal lineage. CRISPR/Cas9-mediated loss knock-out of Rnf149-r function impairs atrial siphon muscle morphogenesis, and down-regulates Tbx1/10 and Ebf, two key determinants of the pharyngeal muscle fate, while upregulating heart-specific gene expression. These phenotypes are reminiscent of loss of FGF-MAPK signaling in the cardiopharyngeal lineage, and integrated analysis of lineage-specific bulk RNA-seq profiling of loss-of-function perturbations identified a significant overlap between FGF-MAPK and Rnf149-r targets. However, functional interaction assays suggested the Rnf149-r does not directly modulate the activity of the FGF-MAPK-Ets1/2 pathway. Instead, we propose that Rnf149-r acts both in parallel to the FGF-MAPK signaling on shared targets, as well as on FGF-MAPK-independent targets through (a) separate pathway(s).

Notch inhibition promotes regeneration and immunosuppression supports cone survival in a zebrafish model of inherited retinal dystrophy

Photoreceptor degeneration leads to irreversible vision loss in humans with retinal dystrophies such as Retinitis Pigmentosa. Whereas photoreceptor loss is permanent in mammals, zebrafish possesses the ability to regenerate retinal neurons and restore visual function. Following acute damage, Muller glia (MG) re-enter the cell cycle and produce multipotent progenitors whose progeny differentiate into mature neurons. Both MG reprogramming and proliferation of retinal progenitor cells require reactive microglia and associated inflammatory signaling. Paradoxically, MG in zebrafish models of photoreceptor degeneration fail to re-enter the cell cycle and regenerate lost cells. Here, we used the zebrafish cep290 mutant to demonstrate that progressive cone degeneration generates an immune response but does not stimulate MG proliferation. Acute light damage triggered photoreceptor regeneration in cep290 mutants but cones were only restored to pre-lesion densities. Using irf8 mutant zebrafish, we found that the chronic absence of microglia reduced inflammation and rescued cone degeneration in cep290 mutants. Finally, single-cell RNA-sequencing revealed sustained expression of notch3 in MG of cep290 mutants and inhibition of Notch signaling induced MG to re-enter the cell cycle. Our findings provide new insights on the requirements for MG to proliferate and the potential for immunosuppression to prolong photoreceptor survival.

HSC-independent definitive hematopoietic cells persist into adult life

SummaryThe stem cell theory that all blood cells are derived from hematopoietic stem cell (HSC) is a central dogma in hematology. However, various types of blood cells are already produced from hemogenic endothelial cells (HECs) before the first HSCs appear at embryonic day (E)11 in the mouse embryo. This early blood cell production from HECs, called HSC-independent hematopoiesis, includes primitive and definitive erythromyeloid progenitors that transiently support fetal blood homeostasis until HSC-derived hematopoiesis is established. Lymphoid potential has traditionally been detected in the extra-embryonic yolk sac (YS) and/or embryos before HSC emergence, but the actual presence of lymphoid progenitors at this stage remains unknown. In addition, whether HSCs in the fetal liver are the main source of innate-like B-1a cells has been controversial. Here, using complementary lineage tracing mouse models, we show that HSC-independent multipotent progenitors (MPPs) and HSC-independent adoptive B-lymphoid progenitors persist into adult life. Furthermore, HSCs minimally contribute to the peritoneal B-1a cell pool; most B-1a cells are originated directly from ECs in the YS and embryo and HSC-independent for life. Our discovery of extensive HSC-independent MPP and B-lymphoid progenitors in adults attests to the complex blood developmental dynamics through embryo to adult that underpin the immune system and challenges the paradigm of HSC theory in hematology.

Stromal inflammation is a targetable driver of hematopoietic aging

Hematopoietic aging is marked by a loss of regenerative capacity and skewed differentiation from hematopoietic stem cells (HSC) leading to impaired blood production. Signals from the bone marrow (BM) niche tailor blood production, but the contribution of the old niche to hematopoietic aging remains unclear. Here, we characterize the inflammatory milieu that drives both niche and hematopoietic remodeling. We find decreased numbers and functionality of osteoprogenitors (OPr) and expansion of pro-inflammatory perisinusoidal mesenchymal stromal cells (MSC) with deterioration of the sinusoidal vasculature, which together create a degraded and inflamed old BM niche. Niche inflammation, in turn, drives chronic activation of emergency myelopoiesis pathways in old HSCs and multipotent progenitors (MPP), which promotes myeloid differentiation at the expense of lymphoid and erythroid commitment and hinders hematopoietic regeneration. Remarkably, niche deterioration, HSC dysfunction and defective hematopoietic regeneration can all be ameliorated by blocking IL-1 signaling. Our results demonstrate that targeting IL-1 as a key mediator of niche inflammation is a tractable strategy to improve blood production during aging.

Modeling the Cellular Origin of EVI1 + MLL-AF9-Driven Acute Myeloid Leukemia (AML)

We previously reported that transplantation of hematopoietic stem cells (HSC) expressing a doxycycline (DOX)-regulated AML-associated MLL-AF9 fusion transgene can induce an invasive and chemoresistant disease in mice formed by tumour cells that express the transcription factor EVI1, a known marker of poor prognosis in AML and some solid cancers. To better understand the association of EVI1 and the cellular origin of the disease we analyzed Evi1-IRES-GFP reporter mice (female, n=8, 8-10 weeks old) and found that not only the, quiescent long-term hematopoietic stem cell (LT-HSC : lineage marker-negative (lin -) cKit +Sca1 +(= LSK), CD34 -CD135 -CD150 +CD48 -, 24±3.7%, n=8) compartment, but also more proliferating multipotent progenitors such as MPP1 (LSK, CD34 +CD135 -CD150 +CD48 -, 23±4.6%, n=8), MPP2 (LSK, CD34 +CD135 -CD150 +CD48 +, 6±2.3%, n=8) and MPP3 (LSK, CD34 +CD135 -CD150 -CD48 +, 2±1%, n=8) contain a significant number of cells that express abundant Evi1 ("Evi1 high") at steady state. Notably, we did not observe any significant changes in numbers of Evi1 + cells nor levels of Evi1 mRNA expression in the LT-HSC and MPP1 compartments 5 days after DOX-mediated induction of the iMLL-AF9 fusion. To address the impact of Evi1 on clonogenic growth of iMLL-AF9-expressing LT-HSC, we plated Evi1 high and Evi1 low naïve cells in methylcellulose (MC) and found that upon addition of DOX, Evi1 high cells formed more colonies with an invasive morphology ("type IV") compared to Evi1 low cells (n=11, p<0.05). Immunophenotypically, cells from Evi1 high cell-derived colonies retained a more immature phenotype, reflected by higher cKit +Sca1 + expression (n=11, p<0.05). Plated Evi1 high cells formed Evi1 + colonies whereas Evi1 low lost Evi1 expression (n=11, p<0.05). To address the differential transformation susceptibility in vivo, we transplanted identical numbers of naïve steady-state Evi1 + iMLL-AF9 LT-HSC and MPP1 into irradiated syngeneic recipients. While recipients of Evi1 + MPP1 cells developed an invasive AML earlier than Evi1 + LT-HSC-transplanted mice (n=27, median latency: 96.5 vs. 146.5d, n.s.), very similar disease phenotypes were observed. In contrast, transplants of Evi1 - MPP1 or LT-HSC resulted in a significantly delayed disease induction (n=31, median latency: >200d; LT-HSC: n.s.; MPP1: p<0.05). Although Evi1 + cell-induced disease did present with more extensive organ infiltration by leukemic blasts than Evi1 - AML the phenotypes were similar. We also wondered whether modulation of the HSC compartment by exogenous factors may change Evi1 expression and affect AML induction. We found that 2 days after a single injection (200mg/kg) of recombinant mouse thrombopoietin (mTPO) the number of LT-HSC (n=23; 647 vs. 1165/10 6 lin - cells, p<0.05), but not of MPP1, significantly increased. Similarly, a single application of the synthetic mTPO receptor agonist Romiplostim (RP, 200mg/kg) resulted after 48h in an increase of LT-HSC (n=14; 647 vs. 1459/10 6 lin - cells, p<0.0005). Likewise, a single dose (10mg/kg) of polyinosinic:polycytidylic acid (pI:pC) also significantly increased the number of LT-HSC (n=9; 460 vs. 2300/10 6 lin - cells, p<0.005) but not of MPP1 after 24h. In contrast, 5-Fluorouracil (5-FU; 150mg/kg) did not significantly change the number of LT-HSC and MPP1, 3- and 6-days post-injection. However, only mTPO and RP but not pI:pC or 5-FU significantly increased the fraction of Evi1 high expressing LT-HSC (23 vs. 50%, 23 vs. 49%; n=29, p<0.0001) and MPP1 (22 vs. 47%, 22 vs- 48%; n=29, p<0.0001). Transplantation of identical numbers of iMLL-AF9 LT-HSC and MPP1 isolated 2 days after mTPO application to the donors into irradiated syngeneic recipients resulted in a significantly faster induction of Evi1 + AML than controls (n=19, MPP1: 35 vs. 96.5d, p<0.0001; LT-HSC: 41 vs 146.5d, p<0.0001). Currently ongoing single-cell RNA sequencing experiments of LT-HSC and MPP1 with and without in vivo mTPO stimulation conditionally expressing the iMLL-AF9 fusion should provide some mechanistic insights into increased susceptibility for EVI1 + AML. Our results so far demonstrate that the dynamics of the HSC compartment critically affects the cellular origin and biology of MLL-AF9 driven AML. Disclosures Kurokawa: MSD K.K.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau.

Single Cell Analysis of Adult Human Hematopoietic Stem and Progenitor Cells Identifies a Novel Lymphoid Primed Multipotent Progenitor That Expands in Relapsed Acute Myeloid Leukemia

Background: Hematopoietic stem and progenitor cells (HSPCs) are capable of generating all human blood cells. Although these cells have been extensively evaluated using both sorted (Corces et al. Nat. Genet. 2016) and single cell assays (Pellin et al. Nat. Commun. 2019), there remains significant uncertainty as to the degree of heterogeneity within HSPC subpopulations and their relevance to disease. The phenotypic diversity within HSPCs needs to be better characterized in order to understand the pathogenesis of numerous blood disorders including hematologic malignancies. To address this need, we extensively characterized healthy bone marrow mononuclear cells (BMMCs) with both whole transcriptome analysis (WTA) and surface marker evaluation. We hypothesized that by utilizing concurrent RNA and multiplexed (n=35) surface marker analysis, we could not only improve HSPC clustering but also characterize specific phenotypic states along unique hematopoietic differentiation trajectories. This analysis allowed us to identify new subpopulations within currently defined hematopoietic stem cells (HSCs), multipotent progenitors (MPPs) and lymphoid primed multipotent progenitors (LMPPs). Methods: Three healthy adult BMMC samples were stained with antibody conjugated oligonucleotides (BD Abseq) and analyzed using a nanowell cell capture system (BD Rhapsody). We filtered 8,070 high quality cells for 2,508 HSPCs, myeloid cells, and lymphocyte precursors. These cells were first clustered using matrix factorization in order to identify unique HSPC states. Novel clusters within the HSC, MPP, and LMPP subpopulations were purified using flow cytometry, and functionally evaluated with both in vitro and in vivo assays. Results: The antibody-derived tags (ADTs) obtained from BD Abseq comprised 33 of the most informative features (n=2000) and resulted in more stable clustering as determined by within sum of squares (WSS = 898 versus 934 for mRNA alone for 20 clusters). Additionally, we were able to design a targeted HSPC panel (n=500 genes) with Abseq which identified similar cell clusters compared to the WTA alone and WTA plus ADT data (Rand index = 0.88). HSPC clustering identified the putative hematopoietic stem cell (HSC), a multipotent progenitor (MPP), and a new lymphoid primed multipotent progenitor (LMPP). A new computational sorting strategy was derived to purify these primitive HSPCs, and subsequently validated with flow cytometry. The functional evaluation of these sorted populations revealed that HSC and MPP cells were capable of increased serial replating ability in vitro suggesting enhanced self-renewal capabilities compared to LMPP cells. All three HSPC subpopulations produced gradients of erythroid and myeloid colonies in methylcellulose, and T-cells in T-cell expansion assays in vitro. The HSCs were the only cells that were able to produce long term engraftment in immunodeficient mice after serial transplantations. Additionally, common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP), and megakaryocyte-erythroid progenitor (MEP) clusters that expressed canonical surface markers were identified. The primitive HSPC clusters were converted into a signature matrix using CIBERSORTx (Newman et al. Nat. Biotechnol. 2019), and bulk acute myeloid leukemia (AML) and healthy samples were deconvolved into respective healthy cell clusters. Using multivariate Cox proportional hazard analysis, we found that high levels of MPPs at diagnosis was the strongest feature associated with worse overall survival (HR = 78.46, 95% confidence interval 7.32-828). Interestingly, after performing paired analysis of diagnostic and relapse cases, the HSC and LMPP expanded significantly in relapsed AML after chemotherapy (p < 0.05) whereas the MPP expanded considerably after stem cell transplant but did not reach significance (p = 0.09). Conclusions: In summary, we identified new cell type clusters within previously defined HSC, MPP, and LMPP subpopulations, and unique surface marker associations using combined single cell WTA and surface marker analysis. We were able to purify these primitive HSPCs using these new markers, in addition to classical markers like CD34, CD90, and CD45RA. Importantly, deconvolution analysis provided preliminary insights into their clinical relevance in AML. Disclosures Ediriwickrema: Nanosive SAS: Patents & Royalties. Majeti: BeyondSpring Inc: Membership on an entity's Board of Directors or advisory committees; Circbio inc: Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutics Solutions Inc: Membership on an entity's Board of Directors or advisory committees; Coherus Biosciences: Membership on an entity's Board of Directors or advisory committees; Acuta Capital Partners: Consultancy; Gilead Sciences, Inc: Patents & Royalties.

C-MYC Augments the Proliferation and Survival of Hematopoietic Stem Cells and Multipotent Progenitors to Drive Myeloproliferative Neoplasms

Human genomic studies have identified frequent MYC amplification and copy number gains in myeloid malignancies, and previous studies have shown that MYC plays important roles in survival of Myeloproliferative Neoplasms (MPN) and Acute Myeloid Leukemia (AML) cells. Notably, our recent studies have shown that MYC impairs myeloid cell differentiation and promotes proliferation of myeloid progenitors and AML cells by controlling genomic methylation. However, it is unclear if increased levels of MYC in hematopoietic stem cells (HSCs) and myeloid progenitors is sufficient to provoke the development of MPN or AML and, if so, how this occurs. To addresses these questions we generated Mx1-Cre;Rosa26-LSL-MYC transgenic mouse model that inducibly overexpress MYC following polyinosinic:polycytidylic acid (pIpC) injection and Cre-mediated deletion of loxp-stop-loxp cassette. MYC overexpression was confirmed by qRT-PCR and immunoblot. Complete blood counts (CBC) with differential in the Mx1-Cre +/-;Rosa26-LSL-MYC +/+ mice vs. -MYC +/-or -wild type (WT) littermate mice at week 23 revealed worsening anemia (Hb, 9.6 vs. 16.3 vs. 15.5g/dL, p<0.0001), lymphopenia (73.2 vs. 84.3 vs. 84.5%, p<0.0001), and monocytosis (7.4 vs. 1.8 vs. 0.9%, p=0.0097). Also, bone marrow (BM) cells from the Mx1-Cre +/-;Rosa26-LSL-MYC +/+ mice showed increased monocyte- and granulomonocyte-colony forming potential (CFU-M and CFU-GM), but with limited self-renewal capacity ex vivo (i.e., no CFU after 5 serial plating). Further, inducible MYC overexpression promotes expansion of HSCs (Lin -Sca-1 +cKit + [LSK]), multipotent progenitors (MPPs; LSK CD48 +CD150 -), common myeloid progenitors (CMPs; Lin -Sca1 -cKit +), granulocyte-monocyte progenitors (GMPs; Lin -Sca-1 -cKit +CD34 +FCγR +), and Gr-1/CD11b+ mature myeloid cells, with concomitant reduction of B220+ or CD3+ cells in the BM and spleen. In addition, MYC overexpression provokes splenomegaly (565 vs. 150 vs. 100mg at week 18~22, p<0.0001), extramedullary hematopoiesis with markedly atypical megakaryopoiesis and myeloid preponderance akin to MPN that reduces overall survival (median OS, 157 days vs. not reached vs. not reached, p<0.0001). Collectively, these findings suggest MYC confers enhanced proliferation and survival properties to HSCs and MPPs leading to MPN-like disease. We have shown MYC oncogenic functions in AML cells requires its suppression of TFEB, an mTORC1 regulated bHLH-LZ transcription factor, and that TFEB functions as a tumor suppressor by inducing IDH1/2-TET2 signaling, thus promoting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) conversion in key genes that drive myeloid differentiation and cell death. Similarly, inducible overexpression of MYC in the Mx1-Cre +/-;Rosa26-LSL-MYC +/+ mice significantly reduces the expression of Tfeb, Idh1 and Idh2, and 5hmC levels in both c-Kit + and Cd11b + BM cells. Further, 4-OHT-mediated silencing of Myc in ex vivo cultured BM cells from the Rosa26-CreER T2+/-;Myc fl/fl mice impairs myeloid cell proliferation and robustly induces the expression of Tfeb, Idh1, and Idh2 as well as levels of 5hmC. Finally, inducible TFEB expression in normal 32D.3 myeloid progenitor cells impairs cell proliferation and upregulates 5hmC levels, and these responses are partially reversed by treatment with 2-hydroxyglutarate, an oncometabolite that inhibits 5mC-to-5hmC conversion. Collectively, these findings suggest that the MYC-TFEB-IDH1/2 epigenetic circuit plays a pivotal role in promoting myeloid proliferation to drive the malignant transformation of HSCs to the MPN. Disclosures Kuykendall: Pharmaessentia: Honoraria; Abbvie: Honoraria; Protagonist: Consultancy, Research Funding; Incyte: Consultancy; Blueprint: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria, Speakers Bureau. Komrokji: Agios: Honoraria, Speakers Bureau; Acceleron: Honoraria; Geron: Honoraria; Novartis: Honoraria; Abbvie: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; JAZZ: Honoraria, Speakers Bureau.

The complex landscape of haematopoietic lineage commitments is encoded in the coarse-grained endogenous network

Haematopoietic lineage commitments are presented by a canonical roadmap in which haematopoietic stem cells or multipotent progenitors (MPPs) bifurcate into progenitors of more restricted lineages and ultimately mature to terminally differentiated cells. Although transcription factors playing significant roles in cell-fate commitments have been extensively studied, integrating such knowledge into the dynamic models to understand the underlying biological mechanism remains challenging. The hypothesis and modelling approach of the endogenous network has been developed previously and tested in various biological processes and is used in the present study of haematopoietic lineage commitments. The endogenous network is constructed based on the key transcription factors and their interactions that determine haematopoietic cell-fate decisions at each lineage branchpoint. We demonstrate that the process of haematopoietic lineage commitments can be reproduced from the landscape which orchestrates robust states of network dynamics and their transitions. Furthermore, some non-trivial characteristics are unveiled in the dynamical model. Our model also predicted previously under-represented regulatory interactions and heterogeneous MPP states by which distinct differentiation routes are intermediated. Moreover, network perturbations resulting in state transitions indicate the effects of ectopic gene expression on cellular reprogrammes. This study provides a predictive model to integrate experimental data and uncover the possible regulatory mechanism of haematopoietic lineage commitments.

Context-Dependent Effects of Inflammation On Retina Regeneration

Inflammation is required for the proliferation of Müller glia (MG) into multipotent progenitors (MGPCs) in the injured fish and avian retinas. However, its function in retina regeneration has not been fully understood. Here we investigated the role of inflammation in three different retinal regeneration paradigms in zebrafish (stab-injury, NMDA-injury and insulin treatment). We first show that different types of immune cells and levels of inflammatory cytokines were found in the retinas of these paradigms. Though zymosan injection alone was insufficient to induce MG proliferation in the uninjured retina, immune suppression significantly inhibited MGPC formation in all three paradigms. Enhancing inflammation promoted MGPC formation after stab-injury, while exhibiting a context-dependent role in the NMDA or insulin models. Furthermore, proper levels of inflammation promoted MG reprogramming and cell cycle re-entry after stab- or NMDA-injury, but excessive inflammation also suppressed MG proliferation in the latter model. Finally, while inflammation promoted retinal neuron regeneration after stab-injury, immune suppression surprisingly achieved the best regeneration in the NMDA model. Our study reveals the complex and context-dependent role of inflammation during retinal repair in fish, and suggests accurate inflammation management may be crucial for successful retina regeneration in mammals.