Lineage Fate Research Articles

Microbiota-induced plastic T cells enhance immune control of antigen-sharing tumors.

Therapies that harness the immune system to target and eliminate tumor cells have revolutionized cancer care. Immune checkpoint blockade (ICB), which boosts the anti-tumor immune response by inhibiting negative regulators of T cell activation1-3, is remarkably successful in a subset of cancer patients, yet a significant proportion do not respond to treatment, emphasizing the need to understand factors influencing the therapeutic efficacy of ICB4-9. The gut microbiota, consisting of trillions of microorganisms residing in the gastrointestinal tract, has emerged as a critical determinant of immune function and response to cancer immunotherapy, with multiple studies demonstrating association of microbiota composition with clinical response10-16. However, a mechanistic understanding of how gut commensal bacteria influence the efficacy of ICB remains elusive. Here we utilized a gut commensal microorganism, segmented filamentous bacteria (SFB), which induces an antigen-specific Th17 cell effector program17, to investigate how colonization with it affects the efficacy of ICB in restraining distal growth of tumors sharing antigen with SFB. We find that anti-PD-1 treatment effectively inhibits the growth of implanted SFB antigen-expressing melanoma only if mice are colonized with SFB. Through T cell receptor clonal lineage tracing, fate mapping, and peptide-MHC tetramer staining, we identify tumor-associated SFB-specific Th1-like cells derived from the homeostatic Th17 cells induced by SFB colonization in the small intestine lamina propria. These gut-educated ex-Th17 cells produce high levels of the pro-inflammatory cytokines IFN-γ and TNF-α, and promote expansion and effector functions of CD8+ tumor-infiltrating cytotoxic lymphocytes, thereby controlling tumor growth. A better understanding of how distinct intestinal commensal microbes can promote T cell plasticity-dependent responses against antigen-sharing tumors may allow for the design of novel cancer immunotherapeutic strategies.

Methionine Restriction Reduces Lung Cancer Progression and Increases Chemotherapy Response.

Targeting tumor metabolism through dietary interventions is an area of growing interest, and may help to improve the significant mortality of aggressive cancers, including non-small cell lung cancer (NSCLC). Here we show that the restriction of methionine in the aggressive KRAS /Lkb1- mutant NSCLC autochthonous mouse model drives decreased tumor progression and increased carboplatin treatment efficacy. Importantly, methionine restriction during early stages of tumorigenesis prevents the lineage switching known to occur in the model, and alters the tumor immune microenvironment (TIME) to have fewer tumor-infiltrating neutrophils. Mechanistically, mutations in LKB1 are linked to anti-oxidant production through changes to cystathionine-β-synthase (CBS) expression. Human cell lines with rescued LKB1 show increased CBS levels and resistance to carboplatin, which can be partially rescued by methionine restriction. Furthermore, LKB1 rescued cells, but not mutant cells, show less G2- M arrest and apoptosis in high methionine conditions. Knock-down of CBS sensitized both LKB1 mutant and non-mutated lines to carboplatin, again rescuing the carboplatin resistance of the LKB1 rescued lines. Given that immunotherapy is commonly combined with chemotherapy for NSCLC, we next wanted to understand if T cells are impaired by MR. Therefore, we examined the ability of T cells from MR and control tumor bearing mice to proliferate in culture and found that T cells from MR treated mice had no defects in proliferation, even though we continued the MR conditions ex vivo . We also identified that CBS is most highly correlated with smoking, adenocarcinomas with alveolar and bronchiolar features, and adenosquamous cell carcinomas, implicating its roles in oxidative stress response and lineage fate in human tumors. Taken together, we have shown the importance of MR as a dietary intervention to slow tumor growth and improve treatment outcomes for NSCLC.

Macrophage-derived extracellular vesicles regulate skeletal stem/progenitor Cell lineage fate and bone deterioration in obesity

Obesity-induced chronic inflammation exacerbates multiple types of tissue/organ deterioration and stem cell dysfunction; however, the effects on skeletal tissue and the underlying mechanisms are still unclear. Here, we show that obesity triggers changes in the microRNA profile of macrophage-secreted extracellular vesicles, leading to a switch in skeletal stem/progenitor cell (SSPC) differentiation between osteoblasts and adipocytes and bone deterioration. Bone marrow macrophage (BMM)-secreted extracellular vesicles (BMM-EVs) from obese mice induced bone deterioration (decreased bone volume, bone microstructural deterioration, and increased adipocyte numbers) when administered to lean mice. Conversely, BMM-EVs from lean mice rejuvenated bone deterioration in obese recipients. We further screened the differentially expressed microRNAs in obese BMM-EVs and found that among the candidates, miR-140 (with the function of promoting adipogenesis) and miR-378a (with the function of enhancing osteogenesis) coordinately determine SSPC fate of osteogenic and adipogenic differentiation by targeting the Pparα-Abca1 axis. BMM miR-140 conditional knockout mice showed resistance to obesity-induced bone deterioration, while miR-140 overexpression in SSPCs led to low bone mass and marrow adiposity in lean mice. BMM miR-378a conditional depletion in mice led to obesity-like bone deterioration. More importantly, we used an SSPC-specific targeting aptamer to precisely deliver miR-378a-3p-overloaded BMM-EVs to SSPCs via an aptamer-engineered extracellular vesicle delivery system, and this approach rescued bone deterioration in obese mice. Thus, our study reveals the critical role of BMMs in mediating obesity-induced bone deterioration by transporting selective extracellular-vesicle microRNAs into SSPCs and controlling SSPC fate.

O01 Mapping the cellular and molecular dynamics of mouse and human hair follicles reveals mechanisms of hair growth

Abstract Introduction and aims Tissue homeostasis in continuously renewing organs, such as the skin, rely on orchestrated cellular and molecular dynamics. The hair follicle (HF) is characterized by distinct keratinocytes organized in transcriptionally and functionally distinct concentric HF layers. The anagen progenitor cells (also called germinative layer cells) are located juxtaposed to the dermal papilla and give rise to each of the differentiated layers. Despite this well-known high level of organization and cell-lineage separation, it is still unclear how the germinative layer cells coordinate hair growth and commit to specific lineages. Methods Here, we employed single-cell RNA sequencing, single-molecule mRNA fluorescence in situ hybridization and in vivo lineage tracing to show how the matrix progenitor cells commit and contribute to the HF lineages depending on their position. Furthermore, we developed a four-dimensional live imaging system of the human HF to construct a global map of single-cell dynamics. Results We show that the HF progenitors dynamically relocate in a conveyor-belt-like movement, switch their lineage fate and gradually restrict their potential. This spatiotemporal cell choreography is characterized by a gradual change of expression from Lgr5 in the lower proximal cup, through Dcn, Id3 and Msx1 in the central part, to distal Lef1 expression where the medulla lineage starts. Detailed in situ mRNA staining and mouse clonal lineage tracing show the transcriptional changes and gradual lineage restriction of matrix progenitors in the spatial context. Combining human HF imaging with fluid dynamics theories, we uncovered a spiral-like downward movement of outer root sheath cells, feeding the germinative layer stem cells, and revealed a pulling force induced by the outer root sheath cells as a critical driver for hair growth. Conclusions Taken together, our study uncovered how HF progenitor cells progress in a conveyor-belt-like manner through several transcriptional states and discovered new cellular and molecular mechanisms regulating hair formation.

ETV4 is a mechanical transducer linking cell crowding dynamics to lineage specification.

Dynamic changes in mechanical microenvironments, such as cell crowding, regulate lineage fates as well as cell proliferation. Although regulatory mechanisms for contact inhibition of proliferation have been extensively studied, it remains unclear how cell crowding induces lineage specification. Here we found that a well-known oncogene, ETS variant transcription factor 4 (ETV4), serves as a molecular transducer that links mechanical microenvironments and gene expression. In a growing epithelium of human embryonic stem cells, cell crowding dynamics is translated into ETV4 expression, serving as a pre-pattern for future lineage fates. A switch-like ETV4 inactivation by cell crowding derepresses the potential for neuroectoderm differentiation in human embryonic stem cell epithelia. Mechanistically, cell crowding inactivates the integrin-actomyosin pathway and blocks the endocytosis of fibroblast growth factor receptors (FGFRs). The disrupted FGFR endocytosis induces a marked decrease in ETV4 protein stability through ERK inactivation. Mathematical modelling demonstrates that the dynamics of cell density in a growing human embryonic stem cell epithelium precisely determines the spatiotemporal ETV4 expression pattern and, consequently, the timing and geometry of lineage development. Our findings suggest that cell crowding dynamics in a stem cell epithelium drives spatiotemporal lineage specification using ETV4 as a key mechanical transducer.

B7H3 (CD276) regulation of myeloid cell differentiation in pulmonary fibrosis

Abstract Myeloid-derived suppressor cells (MDSCs) are induced in cancer and fibrosis, while absent in healthy tissues. The expansions of MDSC are correlated with disease severity and lung function deterioration in the latter. B7H3 induced in lung fibrosis causes the recruitment of MDSCs. However, the mechanism underlying the biogenesis of MDSCs is unclear. To study if B7H3-induced expansion of MDSCs is due to inhibition of the differentiation of myeloid progenitors, the role of B7H3 in the regulation of BM-derived myeloid cells was studied. The results showed that B7H3 deficiency in BM LysM+ myeloid cells significantly increased differentiation to mature myeloid cells. In contrast, treatment with B7H3 inhibited such differentiation. In mice, LysM+ cells-specific B7H3 deficiency induced mature myeloid cell differentiation, but significantly decreased immature MDSCs, which was associated with less immunosuppressive capability. Lack of B7H3 caused a significant reduction of MDSC marker genes characteristic. Furthermore, the delayed B7H3 antibody treatment or by ablating of its secretion from collagen-expressing cells reduced BM-derived MDSCs and PD1-expressing progenitors but increased mature myeloid cell number, which was accompanied by attenuated lung fibrosis. These findings suggested that activated fibroblasts in lung fibrosis may be a key source of soluble B7H3 essential for the signal to BM skewing myeloid lineage fate and induced biogenesis of MDSC

TET2 mutation as prototypic clonal hematopoiesis lesion

Loss of function TET2 mutation (TET2MT) is one of the most frequently observed lesions in clonal hematopoiesis (CH). TET2 a member TET-dioxygenase family of enzymes that along with TET1 and TET3, progressively oxidize 5-methyl cytosine (mC) resulting in regulated demethylation of promoter, enhancer and silencer elements of the genome. This process is critical for efficient transcription that determine cell lineage fate, proliferation and survival and the maintenance of the genomic fidelity with aging of the organism. Partial or complete loss-of-function TET2 mutations create regional and contextual DNA hypermethylation leading to gene silencing or activation that result in skewed myeloid differentiation and clonal expansion. In addition to myeloid skewing, loss of TET2 creates differentiation block and provides proliferative advantage to hematopoietic stem and progenitor cells (HSPCs). TET2MT is a prototypical lesion in CH, since the mutant clones dominate during stress hematopoiesis and often associates with evolution of myeloid malignancies. TET2MT clones has unique privilege to create and persist in pro-inflammatory milieu. Despite extensive knowledge regarding biochemical mechanisms underlying distorted myeloid differentiation, and enhanced self-replication of TET2MT HSPC, the mechanistic link of various pathogenesis associated with TET2 loss in CHIP is less understood. Here we review the recent development in TET2 biology and its probable mechanistic link in CH with aging and inflammation. We also explored the therapeutic strategies of targeting TET2MT associated CHIP and the utility of targeting TET2 in normal hematopoiesis and somatic cell reprograming. We explore the biochemical mechanisms and candidate therapies that emerged in last decade of research.

The role of c-Jun for beating cardiomycyte formation in prepared embryonic body

BackgroundMorbidity and mortality associated with cardiovascular diseases, such as myocardial infarction, stem from the inability of terminally differentiated cardiomyocytes to regenerate, and thus repair the damaged myocardial tissue structure. The molecular biological mechanisms behind the lack of regenerative capacity for those cardiomyocytes remains to be fully elucidated. Recent studies have shown that c-Jun serves as a cell cycle regulator for somatic cell fates, playing a key role in multiple molecular pathways, including the inhibition of cellular reprogramming, promoting angiogenesis, and aggravation of cardiac hypertrophy, but its role in cardiac development is largely unknown. This study aims to delineate the role of c-Jun in promoting early-stage cardiac differentiation.MethodsThe c-Jun gene in mouse embryonic stem cells (mESCs) was knocked out with CRISPR-Cas9, and the hanging drop method used to prepare the resulting embryoid bodies. Cardiac differentiation was evaluated up to 9 days after c-Jun knockout (ko) via immunofluorescence, flow cytometric, and qPCR analyses.ResultsCompared to the wild-type control group, obvious beating was observed among the c-Jun-ko mESCs after 6 days, which was also associated with significant increases in myocardial marker expression. Additionally, markers associated with mesoderm and endoderm cell layer development, essential for further differentiation of ESCs into cardiomyocytes, were also up-regulated in the c-Jun-ko cell group.ConclusionsKnocking out c-Jun directs ESCs toward a meso-endodermal cell lineage fate, in turn leading to generation of beating myocardial cells. Thus, c-Jun plays an important role in regulating early cardiac cell development.

Abstract C133: BBT-4437, a novel, brain-penetrable, reversible pan-TEAD inhibitor targeting the Hippo signaling pathway in solid tumors

Abstract The Hippo pathway regulates several cellular functions including cell lineage fate, proliferation, migration, apoptosis, maintenance of cell stemness and dedifferentiation, and organ size. TEA domain family members 1-4 (TEAD1-4) are evolutionarily conserved transcription factors and core components of the Hippo pathway. TEADs are functional partners of the oncoproteins YAP (Yes-associated protein) and its paralog TAZ (transcriptional co-activator with PDZ-binding motif). Mutations in components of the Hippo pathway have been implicated in several diseases, including cancer. YAP/TEAD activation has also been associated with resistance to targeted therapies by EGFR TKIs in EGFR-mutant NSCLC. Targeting TEAD transcription activity has emerged as a promising approach to addressing cancers with abnormalities in the Hippo pathway. BBT-4437 is a novel, brain-penetrable, reversible pan-TEAD inhibitor that exhibits excellent selectivity in blocking TEAD transcriptional activity in the TEAD reporter gene assay. It demonstrates nanomolar potency in inhibiting cell proliferation in NF2-deficient mesothelioma cells in vitro. BBT-4437 also inhibits the expression of downstream YAP-TEAD target genes (CCN1/CYR61, CCN2/CTGF and IGFBP3) in a concentration-dependent manner in vitro. Moreover, BBT-4437 shows synergistic effect in combination with osimertinib in the H1975 NSCLC cell line. Furthermore, daily oral administration of BBT-4437 leads to significant tumor regression in NF2-deficient H226 and H2373 mesothelioma xenograft models. BBT-4437 demonstrates favorable pharmacokinetic and ADME profiles, including excellent brain penetration. Therefore, BBT-4437 has the potential to be a best-in-class TEAD inhibitor for targeting tumors with aberrant Hippo signaling and overcoming drug resistance caused by upregulation of the Hippo pathway in the clinic. Citation Format: Leila Revollo, Jehrod Brenneman, Sharad Paudyal, Toan Nguyen, Yong-Hee Lee, Taiguang Jimmy Jin. BBT-4437, a novel, brain-penetrable, reversible pan-TEAD inhibitor targeting the Hippo signaling pathway in solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C133.

Extracellular Vesicle Trafficking Regulates Hematopoietic Stem and Progenitor Cell Fitness

Among regulatory pathways critical to hematopoietic stem and progenitor cell (HSPC) function, sphingolipid metabolism has recently been recognized for its role in safeguarding essential HSPC properties of self-renewal and lineage fate. In particular, de novo ceramide (Cer) synthesis appears to regulate a coordinated stress response that results in enhanced self-renewal of human HSPCs. However, the fundamental molecular mechanisms underlying these regenerative stress responses have yet to be uncovered and broadly validated. Moreover, alternative synthetic pathways regulating Cer and sphingomyelin (SM) levels may serve additional regulatory roles in HSPCs. Here we target neutral sphingomyelinase-2 (nSMase2/SMPD3)-dependent SM hydrolysis through pharmacologic (GW4869) and genetic (Cas9-mediated) interruption, and similarly confirm an adaptive ATF4-mediated integrated stress response (ISR). Functionally, transient inhibition led to durable improvements in HSPC reconstitution after primary (>8 fold at steady-state, p<0.001) and secondary (>20 fold at steady-state, p<0.001) transplantation. Surprisingly, lipidomic profiling of GW4869-treated HSPCs demonstrated neither global Cer depletion nor SM accumulation, suggesting an alternative mechanism underlying the observed gains in HSPC fitness. Sphingolipid metabolism also regulates the biogenesis of select subclasses of secreted extracellular vesicles (EVs). To segregate metabolic regulation of HSPCs by sphingolipids from their role in EV formation and secretion, we began to systematically modulate and compare several core EV biogenesis pathways by Cas9/ribonucleoprotein-mediated knockout (KO) of SMPD3, CD63 and TSG101 in HSPC ex vivo expansion cultures . Similar to pharmacologic nSMase2 inhibition, loss of all 3 genes suppressed EV secretion, whereas only depletion of SMPD3 or ESCRT protein TSG101, but not the tetraspanin protein CD63, led to ISR activation in HSPCs (p<0.05). As a functional corollary, clonogenicity and long-term transplantation outcomes were enhanced by KO of SMPD3 (1.5-fold; p<0.01)and TSG101 (2-fold; p<0.001), but reduced after CD63-KO (0.6-fold; p<0.05). One function of EVs is the export of protein and nucleic acid cargo from the cell. We reasoned the observed differences in stress response and HSPC fitness may reflect uniquely altered secretomes. Quantitative proteomic analysis of the vesicle secretome across KO cells revealed impaired secretion of mitochondrial (n=160, FDR=8.3E-22) and oxidative stress-related (n=82, FDR=2.8E-12) proteins from SMPD3- and TSG101- KO, but not CD63-KO HSPCs, highlighting the heterogeneity and specificity of EV cargo trafficking. We validated this observation by pharmacologic blockade of SMPD3 with GW4869, which similarly led to transient decreases in mitochondrial DNA (0.36-fold, p<0.01) and protein (0.6-fold, p<0.05) export. Finally, we confirmed that impaired mitochondrial cargo secretion into EVs (mtEV) after SMPD3 or TSG101, but not CD63, depletion led to transient accumulation of mitochondrial-sourced reactive oxygen species (p<0.01) that specifically triggered the observed ISR activation and downstream mTOR inhibition. Our study reveals a novel mechanism by which secretory cargo trafficking into EVs guards HSPC homeostasis and fitness. We further demonstrate that transient modulation of this pathway, wherein EV cargo retention activates a molecularly-defined stress response pathway, can durably improve long-term transplantation outcomes. Sphingolipid biology inextricably links cellular metabolism and EV biogenesis, making it likely that prior descriptions of Cer-dependent adaptive stress response involve modulation of EV secretion. Finally, our work underscores the high degree of specificity that governs EV secretion in regulating intrinsic cell function. In aggregate, we show that differential EV biogenesis pathways regulate HSPC fate and fitness, with important translational implications in safeguarding stemness and regenerative capacity during ex vivo expansion and therapeutic transplantation.

ZBTB7A Loss Promotes Synthesis of Lipids and Ketone Bodies in Myeloid Leukemia

Cancer cells reprogram their metabolism at many levels to meet the high demand for ATP and metabolites necessary for rapid tumor growth. We recently showed that inactivation of ZBTB7A, a transcription factor essential for lineage fate decision, causes upregulation of glycolysis culminating in increased energy supply in myeloid leukemia cells (Redondo Monte et al., 2020, Oncogene). ZBTB7A mutations are specifically associated with AML t(8;21), pointing toward cooperation with the RUNX1-RUNX1T1 fusion. However, the underlying mechanism is still not fully understood. Besides alteration of glycolysis, we found genes related to fatty acid metabolism upregulated in the K562 ZBTB7A knockout (KO) myeloid leukemia cell line. This led us to hypothesize that the loss of ZBTB7A could provide the cells with additional energy via increased fatty acid oxidation. To further investigate the role of ZBTB7A in fatty acid metabolism we performed metabolic flux (Seahorse, Agilent) and metabolic tracing experiments. In the palmitate oxidation assay, using a setting where the cells were cultivated in a substrate-limited medium overnight prior to the experiment and deprived of glutamine during the assay, the K562 ZBTB7A KO cells did not exhibit an advantage in oxidizing this substrate for ATP production. Moreover, in this context, the ZBTB7A KO cells responded negatively to the inhibition of the CPT1A enzyme by Etomoxir, suggesting that these cells are not dependent on fatty acid oxidation. Interestingly, RNA-seq revealed that in K562 ZBTB7A KO cells several genes from the electron transport chain (ETC) were downregulated, supporting the idea that these cells do not depend on OXPHOS metabolism. Metabolic tracing of the 13C6 glucose isotope by mass spectrometry confirmed that K562 ZBTB7A KO cells exhibit upregulated glycolysis and pentose phosphate pathways, as previously reported (Redondo Monte et al., 2020, Oncogene; Liu et al., 2014, Genes Dev.). Conversely, tricarboxylic acid cycle (TCA) metabolites were not different between control and knockout cells. We observed that upregulation of glycolysis resulted in high amounts of acetyl-CoA, which is not entering the OXPHOS metabolism but is used as a precursor for palmitate synthesis instead (Figure 1A). Of note, palmitate is required for the production of several complex lipids in cell membranes, which are highly demanded in cancer cells. Accordingly, the transcript level of FASN, the enzyme that catalyses palmitate synthesis, is upregulated in core binding factor (CBF) AML patients with mutation in ZBTB7A (Figure 1B). Remarkably, the K562 ZBTB7A KO cells preferentially produce acetoacetate (ketone bodies) from the additional acetyl-CoA and secrete it to the medium (Figure 1A). The genes HMGCS1 and ACAT1, encoding enzymes that enable ketone bodies reutilization in lipid synthesis, are downregulated upon ZBTB7A loss, indicating that these cells do not use the ketone bodies. Since ketone bodies not only serve as a substrate for ATP production but also act as signaling mediators, we hypothesize that ketone bodies might fuel the bone marrow microenvironment to support leukemia cells expansion. In summary, we have advanced the understanding of ZBTB7A loss in leukemia metabolism, showing increased lipid and ketone bodies synthesis at the metabolite level. Lipid synthesis enzymes, such as FASN, FADS1, and FADS2 are attractive drug targets for therapeutic applications, especially in combination with standard therapies and glycolysis inhibitors.

DNMT3A Regulates Megakaryocyte-Primed HSC Lineage Fate Decisions

The de novo DNA methyltransferase DNMT3A is important for normal hematopoiesis and hematopoietic stem cell (HSC) differentiation. Mutations in DNMT3A in humans lead to clonal expansion and a higher risk of malignancy development. In mice, ablation of DNMT3A enhances self-renewal and inhibits differentiation. Given that the pool of HSCs is heterogeneous, we considered whether DNMT3A loss preferentially affected particular HSC sub-types and hypothesized a role in megakaryocyte (Mk)-primed HSCs, given their position at the very top of the HSC hierarchy. To examine this, we utilized Dnmt3a+/- mice in which Mk-primed HSCs are labeled with GFP due to an insertion of GFP under the von Willebrand factor (VWF) promoter (Sanjuan-Pla et al. 2013, Nature). This strain also facilitates the tracing of GFP pos megakaryocytes (Mks) and platelets in Dnmt3a+/- mice. We performed competitive transplantation with HSCs that were either GFP pos (Mk-primed) or GFP neg from WT and Dnmt3a+/- mice. The Dnmt3a+/- GFP pos HSCs exhibited significantly lower engraftment than the GFP neg conditions at 6 weeks post-transplant (Fig. A). While Mk-primed HSCs exhibited delayed lymphoid contribution, which was exacerbated in the Dnmt3a+/- condition, lymphoid contribution normalized in all groups by 18 weeks post-transplant. Strikingly, the Dnmt3a+/- GFP pos HSCs displayed significantly higher platelet contribution than all other groups relative to their engraftment without altering total platelet counts. We also performed competitive transplantation with WT or Dnmt3a+/- GFP pos whole bone marrow. Upon secondary transplant, the Dnmt3a+/- cells exhibited significantly higher overall contribution to blood production. Additionally, the Dnmt3a+/- bone marrow displayed a marked myeloid/Mk/platelet bias relative to engraftment (Fig. B), and a significantly higher percentage of the HSC compartment was Mk-primed, although the total progenitor pool was smaller. It is likely that the enhanced number of Mk-primed HSCs in the Dnmt3a+/- secondary transplant condition contributes to increased myeloid/Mk/platelet skewing but that overall engraftment is rescued by the presence of GFP neg HSCs. We next examined whether the differences observed in the HSC vs whole bone marrow transplant models were due to transplant stress or were present in the germline Dnmt3a+/- animals. Although we observed a slight increase in the number of Mks in the bone marrow in the germline Dnmt3a+/- animals, their Mks were the same size as WT. Their Mk ploidy, megakaryocyte-erythroid progenitor (MEP) composition, and %GFP pos HSCs were similar to littermate controls. However, while WT GFP pos HSCs were exceptionally enriched in the portion of HSCs with the highest stemness potential (marked by high Hoechst efflux and high CD150 expression), this enrichment was abrogated in Dnmt3a+/- GFP pos HSCs. Overall, these data indicate that megakaryocyte and platelet production is augmented from Dnmt3a+/- HSCs, likely due to shifts in the composition of the HSC pool, which are exacerbated after transplantation. To determine whether DNMT3A loss affects Mk and platelet function, we cultured bone-marrow-derived Mks and isolated platelets from mouse blood. Dnmt3a+/- Mks formed proplatelets at similar rates to WT. Dnmt3a+/- platelets activated and aggregated in response to agonists, although at the gene and protein expression levels, several coagulation factors and key cytoskeletal components were downregulated. Finally, we subjected WT and Dnmt3a+/- MEPs and Mks to enzymatic DNA methyl-sequencing to examine how genome-wide methylation patterns are altered during the differentiation trajectory in the WT and Dnmt3a+/- contexts. Our findings demonstrate that DNMT3A plays an important role in the lineage fate decisions of Mk-primed HSCs and implicates DNA methylation in Mk differentiation. Our data suggest that platelets generated from DNMT3A-mutant Mks may be over-represented in the blood of individuals with clonal hematopoiesis (CH) relative to apparent clone size. Given the association between CH and increased risk of cardiovascular disease and stroke, the role of DNMT3A-loss in Mk and platelet biogenesis and function warrants further investigation.

BCR-ABL1 Dislocates NPM1, PU.1 and KLF1 into Cytoplasm to Thereby Skew Granulo-Monocytic and Impede Erythroid Differentiation

BCR-ABL1 causes chronic myeloid leukemia (CML) but the molecular mechanisms by which it skews granulo-monocytic commitment towards granulocytes, increases erythroid precursors yet causes anemia (Bruns et al, Leukemia 2009), and positions for specific lineage-phenotypes of blast crises (granulocytic, erythroid, B-lymphocytic), are unknown. To investigate these mechanisms, we reasoned that distinctly altered myeloid differentiation suggests specific impacts of BCR-ABL1 on master transcription factors (MTFs) that specify and drive myeloid lineage fates. Although wildtype ABL1 is nuclear, BCR-ABL1 is cytoplasmic. We therefore examined cellular location of lineage MTF in CML blast crisis cells K562 and LAMA84. These cells, like 7/14 (50%) of CML cell lines in the CCLE repository, highly express the lineage MTFs GATA1 and KLF1, that specify and drive to terminal erythroid fates. Incongruously, K562 and LAMA84 also highly express PU.1 essential for monocyte or B-cell fates. Using immunofluorescence microscopy (IF) of methanol-fixed K562 ( Fig.1A) and LAMA84, we found GATA1 in nuclei, but KLF1 and PU.1 were in cytoplasm. We corroborated these results by Western blot for these MTF in nuclear vs cytoplasmic fractions. Kinase functions of ABL1 in BCR-ABL1 are essential for BCR-ABL1 mediated oncogenesis. Therefore, we investigated if these kinase functions mediate KLF1 and PU.1 dislocation: one hour (1h) exposures to ABL1-kinase inhibitors imatinib 250nM or dasatinib 1nM relocated KLF1 and PU.1 into nuclei, seen both by IF ( Fig.1A) and by Western blot of nuclear/cytoplasmic fractions. We then examined cell fate impacts of the KLF1 and PU.1 nuclear relocations. Imatinib and dasatinib upregulated terminal erythroid lineage-differentiation markers transferrin (CD71) or glycophorin A (CD235a) (~10-fold increase in median fluorescence intensity, flow cytometry at 72h) ( Fig.1B), and increased hemoglobinization, evident by cell pellet reddening to the naked eye. The monocyte differentiation marker CD14 was not increased. As expected from resumed lineage-maturation, proliferation measured at 72h was terminated by imatinib 250nM or decreased ~50% by dasatinib 1nM vs vehicle, even though these drug concentrations did not increase apoptosis vs vehicle, measured by annexin staining at 24h. To further investigate the pathway by which BCR-ABL1 kinase dislocates KLF1 and PU.1 into cytoplasm, we then looked at NPM1, because: (a) NPM1 chaperones PU.1, and mutant-NPM1 that causes acute myeloid leukemia (AML) dislocates into cytoplasm, dislocating PU.1 with it (Gu et al, J Clin Invest 2018); and (b) NPM1 is neither mutated nor deleted (by 5q-) in CML blast crises, even though such alterations are highly recurrent in AMLs, consistent with NPM1 participating in the BCR-ABL1 pathway to begin with. NPM1 was accordingly mostly in cytoplasm in K562 and LAMA84 cells, and 1h of imatinib or dasatinib relocated NPM1 into nuclei, with NPM1/KLF1 colocalizing ( Fig.1A). These results were corroborated by Western blot of nuclear/cytoplasmic fractions. We are presently immunoprecipitating endogenous NPM1 from nuclear/cytoplasmic fractions of K562 cells and analyzing by mass spectrometry (LCMS/MS) to examine: (a) NPM1 chaperone function for KLF1 as well as PU.1; and (b) NPM1 phosphorylation in the different fractions and kinase-inhibitor impacts on this. Taken altogether, these results outline a pathway in which BCR-ABL1 phosphorylates NPM1 to dislocate it into cytoplasm, to hence also dislocate chaperoned KLF1 and PU.1. Interference with specific lineage MTFs in this way can: (a) expand erythroid precursors but block their maturation, because nuclear GATA1 allows erythroid commitment, but KLF1 (in nuclei) is needed for maturation; (b) skew commitment decisions to granulocytes over monocytes, via decreased nuclear PU.1/CEBPA ratio; (c) expand mature granulocytes, because PU.1 contributes to, but is not essential for, granulocyte lineage-maturation, and NPM1 is downregulated in the granulocyte lineage, releasing terminal maturation; (d) transform B-cell lineage precursors, since PU.1 is an essential B-cell MTF; and (e) position for specific lineage-phenotypes and genetics of CML blast crises, evolving from these foundations. Since the ABL family kinase ABL2 is amplified in a third of all cancers, these motifs for altering lineage MTF function for oncogenesis may operate beyond CMLs.

Biliverdin Reductase B Protects Against Primaquine Induced Hemolysis - Identification of a Novel Anti-Oxidant Pathway in Murine Erythrocytes

Background: Biliverdin Reductase B (BlvrB) is the fourth most abundant enzyme in red blood cells (RBCs) and is widely expressed in other tissues. In normal heme catabolism, porphyrin is converted to biliverdin-alpha (by hemoxygenase (HO)) and then reduced to bilirubin-alpha (by biliverdin reductase A (BlvrA)). Bilirubin-alpha is then glucuronidated and excreted. The large body of literature investigating heme metabolism, bilirubin-based pathologies, and the oxidative biology of bilirubin/biliverdin in adults focuses almost exclusively on BlvrA and the alpha isomers of biliverdin and bilirubin. However, porphyrin is a tetrapyrrole with 4 distinct meso-carbon bridges, each of which can be broken to generate a distinct biliverdin isomer (alpha, beta, gamma, or delta). Although HO is exquisitely specific for the alpha-bond (generating only biliverdin-alpha), chemical coupled oxidation of heme yields all four biliverdin isomers. BlvrB (and not BlvrA) can reduce each of non-alpha biliverdin isomers into their corresponding bilirubin isomers (bilirubin-beta is prevalent in neonatal jaundice and kernicterus). BlvrB also reduces flavins and additional quinone metabolites. Despite BlvrB's abundance and evolutionary conservation, its only known function is a subtle effect on hematopoietic lineage fate (megakaryocyte vs. erythroid). BlvrB has no known function in mature RBCs. We hypothesized that BlvrB plays an important antioxidant role that would only become obvious when other redundant anti-oxidant pathways were limited. Methods: A novel BlvrB knockout mouse was generated on a C57BL/6 background. A model of primaquine (PQ) induced oxidative hemolytic anemia in G6PD deficient mice was used as a clinically relevant scenario in which compromised antioxidant biology leads to treatment limiting pharmacotoxicity in RBCs. BlvrB-KO mice were crossed with two strains of mice that had murine G6PD replaced with either the A- deficient variant of human G6PD (hG6PD(A-)) or the non-deficient form of human G6PD (hG6PD(ND)). Four mouse strains (i.e., hG6PD(ND) and hG6PD(A-) mice with or without BlvrB deletion) received either a 4-day course of PQ or control PBS injections. Anemia was monitored by hematocrit (HCT). Reticulocyte count (RET) was monitored by flow cytometry as CD71+, thiazole orange+ erythrocytes. Peripheral blood was subjected to high resolution metabolomics to elucidate biochemical changes. Results: All mouse strains were healthy at baseline, with normal measured hematological parameters (i.e., Hct 40, RET 1.5%). No change in HCT or RET was observed in hG6PD(ND) or BlvrB-KOxhG6PD(ND) mice receiving PQ or in any strain receiving PBS. PQ induced hemolytic anemia in hG6PD(A-) mice, with an average HCT of 32.7 (18% drop) with RET of 3.1%. A significant exacerbation of PQ induced hemolysis was observed in BlvrB-KOxhG6PD(A-) mice that had an average HCT of 22.5 (44% drop) with RET of 5.3%. (n= 11 mice per group, p <0.001 between groups). Blood from PQ-treated hG6PD(A-)xBlvrBKO mice showed defective glycolysis at the level of glyceraldehyde 3-phosphate dehydrogenase, increased levels of multiple long-chain acylcarnitines, altered redox metabolism, and specific increases of hypoxanthine and sphingosine-1-phosphate (S1P). Three separate experiments were performed with similar results. Discussion: These data demonstrate that BlvrB deletion exacerbates oxidative injury and suggest that BlvrB provides a novel and hitherto undescribed antioxidant pathway in RBCs. Because approximately half a billion humans exhibit some form of G6PD deficiency, these data are also directly relevant to a large population of humans, which were modeled using hG6PD(A-) mice. These studies also define the metabolic lesion in peripheral blood. For example, GAPDH is well known to be highly sensitive to inactivation by oxidative damage, resulting in impaired glycolysis. Changes in hypoxanthine and S1P are known to be associated with decreased RBC function and survival. Finally, altered carnitine metabolism is associated with repair of lipid peroxidation in RBCs via the Lands Cycle. Although the mechanism by which BlvrB facilitates antioxidation is not yet clear, we hypothesize it involves redox cycling of non-alpha isomers of biliverdin and bilirubin. However, the mechanisms may be through reduction of flavins and/or other endogenous substrates. Ongoing studies are testing these hypotheses.

The Effect of SCD-1 Inhibition on Human Hematopoietic Stem Cell Mitochondrial Metabolism, Cell Proliferation, and Differentiation Potential

Hematopoietic stem cells (HSC) give rise to all blood lineages and sustain the production of blood cells throughout life. Due to their inherently high regenerative potential, HSC are used in a variety of clinical settings, including bone marrow transplantation (BMT) directly to cure a variety of hematologic and oncologic disorders often with gene therapy. During regeneration, HSC are activated into cycle. For this, HSC undergo drastic mitochondrial and metabolic remodeling to meet the bioenergetic and biosynthetic needs of activated HSC. A growing body of evidence indicates that HSC sustain injury during activation. This remodeling is important for optimal HSC function but is permanently changed after HSC activating stress. It is thus important to identify the metabolic needs of activated HSC to improve HSC functions for therapeutic purposes. In both murine and human HSC, numerous metabolic enzymes get upregulated during activation, including those involved in de novo lipid synthesis. This metabolism is poorly understood in human HSC. Our overarching question is to understand the metabolic needs of HSC within the human system. Stearoyl-co-A-desaturase 1 (SCD-1), an enzyme responsible for conversion of stearic to oleic acid within the de novo lipid synthesis pathway, is suspected to play a role in metabolic reprogramming after stress. Using CD34+ mobilized human peripheral blood, we analyzed the effect of SCD-1 inhibition on HSC and progenitor proliferation, mitochondrial metabolism, and lineage differentiation potential. Cells were cultured either in vehicle control conditions or with SCD-1 inhibitor (SCDi). Subsequently, cells were counted and analyzed by flow cytometry on Days 4-5 and 10-15. More primitive or stem cell markers were utilized on Days 0 and 5 whereas differentiation markers were utilized on Day 10-15. Both tetramethylrhodamine-ethyl ester dye (TMRE) and MitoSOX reagents were used to assess mitochondrial membrane potential and mitochondrial ROS, respectively. Our results demonstrate that the size of the HSC population generated by SCDi-treated CD34+ cells within 5 days of culture was similar to vehicle treated cells and was composed of similar proportion of CD34+CD38+, CD34+CD38-, and CD38+CD34- cells, indicating that SCD-1 inhibition does not alter HSC expansion under these culture conditions. However, TMRE levels were lower in all HSC populations whereas mitoSOX levels were unchanged by SCD-1 inhibition compared to vehicle. In differentiation conditions, SCDi cultures were composed of a larger proportion of myeloid progenitors and a smaller proportion of erythroid cells after 10-15 days, compared to vehicle-treated cultures. These findings suggest that de novo lipid synthesis is necessary for HSC differentiation but dispensable for proliferation/expansion in vitro. We then used a xenotransplant model to assess the effect of SCDi on HSC regenerative potential in vivo. CD34+ mobilized human peripheral blood cells were cultured for 3 days under conditions that maintain HSC functions in vitro and transplanted into immunodeficient, sublethally irradiated NSG mice. Peripheral blood of xenotransplanted mice was analyzed monthly for human cell chimerism and mature blood lineage potential from 1 to 6 months post-transplant. Bone marrow analysis was performed at the 6 month time point as well, assessing for both HSC markers and lineage markers. Mice that received SCDi-treated cells exhibited human cell chimerism at a level similar to mice that were transplanted with vehicle treated cells, at about 3 to 30%. However, SCDi-treated cells gave rise to a lymphoid-biased graft, in particular T cells, both in the peripheral blood and in the bone marrow of xenotransplanted mice, compared to a more balanced myeloid-lymphoid graft from vehicle treated cells. In conclusion, these findings suggest that de novo lipid synthesis is critically important for HSC lineage fate and balanced differentiation in vitro and in vivo. This is especially clinically relevant as this work may implicate a possible therapeutic target as improving de novo lipid synthesis may aid in patients who suffer from persistent cytopenias after BMT and graft failure.

Title: MLLT1 Is Required to Maintain Normal B-Lineage Hematopoiesis and Mitochondrial Function

MLLT1 (aka ENL) is an epigenetic reader protein that binds to crotonylated/acetylated lysine residues of histone 3 through its N-terminal YEATS domain. Multiple different positive and negative regulatory complexes compete for binding to the MLLT1 C-terminal domain. MLLT1 has been implicated in leukemic stem cell survival in MLL-rearranged cell lines but its role in normal hematopoiesis has not been investigated. Mllt1 is expressed in multiple bone marrow cell subsets, with high expression levels in multipotent and B-lineage progenitors. We generated conditional Mllt1 knockout ( Mllt1 del) mice and observed a B-lineage defect in bone marrow of Mllt1 deleted mice compared to control Mllt1 fl/fl mice. Mitochondrial activity has been linked to hematopoietic lineage fate decision choices. We performed seahorse mitochondrial stress test on lineage negative (stem and progenitor) bone marrow cells and found that lineage negative cells of Mllt1 del mice have lower mitochondrial activity (as a measure of oxygen consumption rate) than control Mllt1 fl/fl mice. In addition, we observed mitochondrial mass (as a quantitative measure of ratio of mtDNA gene CytB / nDNA gene ActB) to be decreased in lineage negative cells of Mllt1 del mice when compared to control Mllt1 fl/fl mice. Surface marker labeling was used to sort more defined subpopulations from bone marrow of Mllt1 del and Mllt1 fl/fl mice. We observed decreased mitochondrial mass in MPP4s and MEPs of Mllt1 del mice when compared to control Mllt1 fl/fl mice. Flow cytometry-based cell surface labeling to distinguish different bone marrow progenitors, plus mitochondria staining using MitoTracker Green (for mitochondrial mass) and MitoTracker Red CMXRos (for mitochondrial membrane potential) showed altered mitochondrial function of B-lineage progenitors in Mllt1 del mice when compared to control Mllt1 fl/fl mice. Thus, Mllt1 is essential for normal mitochondrial function, which may be necessary to maintain specific bone marrow hematopoietic subpopulations. Ongoing studies include gene expression profiling of sorted lymphoid progenitor populations and analysis of direct Mllt1 target genes that contribute to the altered phenotype in Mllt1 del mice.

Myelo-Erythroid Lineage Segregation Is Regulated By the GATA-2 Interactome

Hematopoiesis arises from hematopoietic stem cell differentiating into downstream progenitors through a multistep process of sequential lineage potential restriction leading to the final production of >10 hematopoietic cell type.The regulation of hematopoietic lineage development involves synergistic and antagonistic interactions between transcription factors (TF) with lineage-restricted expression patterns, both through protein-protein interaction and direct transcriptional activation.Eosinophils, basophils and mast cells are key myeloid cell types involved in allergic inflammation and have been proposed to arise from a granulocyte-macrophage progenitor (GMP), that also generates neutrophils and macrophages, with GATA-2 up-regulation as a critical determinant. However, recent work identified a distinct basophil/eosinophil/mast cells progenitor (BEMP) that arises from multi-potent erythroid-primed multipotent progenitors (EMPP) where GATA-2 is already expressed ( Drissen et al., 2016). Here, we show that the GATA-2 interactome, rather than GATA-2 expression, controls myeloid lineage choice in EMPPs. To investigate the molecular mechanism instructing lineage decision in EMPP, we performed gene-expression profiling (RNA-seq) comparing its downstream progeny, early restricted erythroid/megakaryocyte progenitor (preMegE) and BEMP. This analysis identified differential expressed genes, including antagonist gene-expression pattern between the LIM-only protein 2 ( Lmo2) and protein 4 ( Lmo4), both previously shown as GATA-2 interactor. While Lmo2 function in hematopoiesis have been exhaustively studied, the role of Lmo4 remains unknown. In colony forming unit (CFU)-assay, overexpression of Lmo4 in EMPP mediated by lentiviral delivery, resulted in an increased number of mast-colonies associated with a decreased of erythroid/megakaryocyte colonies, while the total colony number was unchanged. In addition, transplantation of hematopoietic lineage-negative cells transduced with Lmo4/empty(Ctrl) lentiviral particles, showed an increased frequency of basophils and eosinophils associated with a decrease of erythroid cells in Lmo4 overexpressing cells compared to Ctrl, in both bone marrow and spleen of recipient mice, 8 weeks after transplantation. Analysis of transduced Lineage-negative Kit + (LK) compartment of recipient mice, also revealed an increased frequency of BEMP associated with a decreased frequency of preMegE in Lmo4overexpressing cells compared to Ctrl. Taken together, these data suggested that Lmo4 overexpression instruct lineage decision of EMPP toward basophil/eosinophil/mast lineages.Over-expression followed by co-immunoprecipitation in HEK-293T cell line revealed that LMO4 compete with LMO2 binding to GATA-2, suggesting a mutually exclusive transcriptional complex. More importantly, lack of interaction between LMO4 and GATA-2 mediated by a point mutation in the zinc-finger 1 domain (ZnF1) of GATA-2 (GATA2-G320D), frequently identified in AML/AEL, resulted in an absence of mast lineage potential of EMPP compared to GATA2 +/+. Moreover, over-expression of Lmo4 in EMPP from GATA2 G320D/G320D mice compared to GATA2 +/+, failed to instruct myeloid fate in vitro. Overall, our data identified a specific molecular mechanism of hematopoietic cell fate decision driven by protein-protein interaction with GATA-2. Perturbation of GATA-2 interactome through over-expression of the cofactor Lmo4 or specific point mutation in GATA2 ZnF1 domain results in lineage fate switch of EMPP toward the erythroid/megakaryocyte or basophil/eosinophil/mast lineages.

Preleukemic single-cell landscapes reveal mutation-specific mechanisms and gene programs predictive of AML patient outcomes

Acute myeloid leukemia (AML) and myeloid neoplasms develop through acquisition of somatic mutations that confer mutation-specific fitness advantages to hematopoietic stem and progenitor cells. However, our understanding of mutational effects remains limited to the resolution attainable within immunophenotypically and clinically accessible bulk cell populations. To decipher heterogeneous cellular fitness to preleukemic mutational perturbations, we performed single-cell RNA sequencing of eight different mouse models with driver mutations of myeloid malignancies, generating 269,048 single-cell profiles. Our analysis infers mutation-driven perturbations in cell abundance, cellular lineage fate, cellular metabolism, and gene expression at the continuous resolution, pinpointing cell populations with transcriptional alterations associated with differentiation bias. We further develop an 11-gene scoring system (Stem11) on the basis of preleukemic transcriptional signatures that predicts AML patient outcomes. Our results demonstrate that a single-cell-resolution deep characterization of preleukemic biology has the potential to enhance our understanding of AML heterogeneity and inform more effective risk stratification strategies.

Systemic administration of STING agonist promotes myeloid cells maturation and antitumor immunity through regulating hematopoietic stem and progenitor cell fate.

STING is a pivotal mediator of effective innate and adaptive anti-tumor immunity; however, intratumoral administration of STING agonists have shown limited therapeutic benefit in clinical trials. The systemic effect of the intravenous delivery of STING agonists in cancer is not well-defined. Here, we demonstrated that systemic administration of STING agonist inhibited melanoma growth, improved inflammatory effector cell infiltration, and induced bone marrow mobilization and extramedullary hematopoiesis, causing widespread changes in immune components in the peripheral blood. The systemically administered STING agonist promoted HSC expansion and influenced lineage fate commitment, which was manifested as the differentiation of HSPCs was skewed toward myeloid cells at the expense of B-cell lymphopoiesis and erythropoiesis. Transcriptome analysis revealed upregulation of myeloid lineage differentiation-related and type I interferon-related genes. This myeloid-biased differentiation promoted the production and maturation of myeloid cells toward an activated phenotype. Furthermore, depletion of Gr-1+ myeloid cells attenuated the anti-tumor immunity of STING agonist. Our findings reveal the anti-tumor mechanism of systemic administration of STING agonist that involves modulating HSPC differentiation and promoting myeloid cells maturation. Our study may help explain the limited clinical activity of STING agonists administered intratumorally.

Anaplastic transformation in thyroid cancer revealed by single cell transcriptomics.

The deadliest anaplastic thyroid cancer (ATC) often transforms from indolent differentiated thyroid cancer (DTC); however, the complex intra-tumor transformation process is poorly understood. We investigated an anaplastic transformation model by dissecting both cell lineage and cell fate transitions using single cell transcriptomes and genetic alteration data from patients with different subtypes of thyroid cancer. The resulting spectrum of ATC transformation included stress-responsive DTC cells, inflammatory ATC cells (iATCs), mitotic-defective ATC cells and extended all the way to mesenchymal ATC cells (mATCs). Further, our analysis identified two important milestones: 1) a diploid stage, where iATC cells were diploids with inflammatory phenotypes, and 2) an aneuploid stage, where mATCs gained aneuploid genomes and mesenchymal phenotypes producing excessive collagens and collagen-interacting receptors. In parallel, cancer-associated-fibroblasts showed strong interactions among mesenchymal cell-types, macrophages shifted from M1 to M2 states, and T cells reprogrammed from cytotoxic to exhausted states, highlighting new therapeutic opportunities for ATC.