Epigenetic and Transcriptional Regulation of Self Renewal in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is diagnosed in >20,000 people/year in the United States alone and is associated with a poor prognosis. AML arises due to altered transcriptional programs resulting from mutations and chromosomal rearrangements. Frequently, this altered transcription is a consequence of epigenetic deregulation. Indeed, over 70% of AML patients harbor mutated epigenetic modifiers, which regulate chromatin accessibility and gene expression. Aberrant expression of the HOXA gene cluster, which can result from epigenetic deregulation, drives transformation of ~50% of AML, including those associated with poor prognosis. One manner in which the HOXA gene cluster becomes aberrantly expressed is through 11q23 chromosomal translocations involving the Mixed Lineage Leukemia 1 (MLL1) gene. These events result in the formation of fusion genes encoding MLL fusion oncoproteins which transcriptionally activate oncogenes, including the HOXA cluster. Our lab and others have demonstrated that the Polymerase Associated Factor complex (PAF1c), an epigenetic regulator complex, interacts directly with and recruits wildtype MLL1 and MLL-fusion oncoproteins to target loci like HOXA9 and MEIS1. The PAF1c-MLL interaction is required for leukemia cell proliferation, but dispensable for normal hematopoiesis. Mutations and aberrant expression of subunits of the PAF1c are observed in various malignancies, suggesting that the PAF1c must be tightly regulated for proper cellular development. However, the biochemical regulation of the PAF1c that allows for its dynamic regulation of gene expression in AML is not fully understood. To better understand the regulation of the PAF1c, we use a proteomics approach to identify novel interaction partners of the PAF1c in AML cells. This study reveals a novel interaction between the PAF1c and the H3K9 methyltransferase SETDB1. The PAF1c-SETDB1 interaction represses the target genes Hoxa9 and Meis1 in murine MLL-AF9 driven leukemic cells and human AML cell lines. SETDB1 mediated transcriptional repression is correlated with an increase in promoter H3K9 trimethylation (H3K9me3). These data suggest that SETDB1 epigenetically represses pro-leukemic gene expression in AML. Therefore, we next explore the biological impact of SETDB1 expression and H3K9 methylation on AML. We note that expression of SETDB1 in AML patient samples is significantly lower compared to normal hematopoietic cells. Further, higher SETDB1 expression correlates with a significantly better overall survival in AML patients. These data are consistent with SETDB1 negatively regulating pro­leukemic genes and suggests that SETDB1 expression and H3K9 methylation levels may be correlated with AML patient prognosis. We demonstrate that overexpression of SETDB1 significantly delays MLL­AF9 mediated leukemogenesis in vivo by inducing differentiation of leukemic cells. We also explore how chemical inhibition of H3K9 methylation affects AML transformation. Treatment with H3K9 methyltransferase inhibitor UNC0638 is antagonistic to established AML cell growth. In contrast, UNC0638 preserves mouse hematopoietic stem and progenitor cells (HSPCs) in culture and increases the amenability of bone marrow cells to be transformed by the MLL-AF9 oncogene. Transcriptome analyses demonstrate that overexpression of SETDB1 downregulates Hoxa and pluripotency gene programs. ChIP-sequencing and ATAC-sequencing of AML cells show that overexpression of SETDB1 leads to the acquisition of a more compact, epigenetically silenced chromatin state at the promoters of genes that are critical for AML, including Dock1 and MLL-AF9 target genes Hoxa9 and Six1, and others. Together, these data reveal a previously unrecognized role for SETDB1 and H3K9 methylation in suppressing AML by epigenetically silencing pro-leukemic target genes and promoting differentiation.PHDMolecular & Cellular PathologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/149783/1/jropa_1.pd

Human Hematopoietic Stem, Progenitor, and Immune Cells Respond Ex Vivo to SARS-CoV-2 Spike Protein

This article is made available for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.Despite evidence that SARS-CoV-2 infection is systemic in nature, there is little known about the effects that SARS-CoV-2 infection or exposure has on many host cell types, including primitive and mature hematopoietic cells. The hematopoietic system is responsible for giving rise to the very immune cells that defend against viral infection and is a source of hematopoietic stem cells (HSCs) and progenitor cells (HPCs) which are used for hematopoietic cell transplantation (HCT) to treat hematologic disorders, thus there is a strong need to understand how exposure to the virus may affect hematopoietic cell functions. We examined the expression of ACE2, to which SARS-CoV-2 Spike (S) protein binds to facilitate viral entry, in cord blood derived HSCs/HPCs and in peripheral blood derived immune cell subtypes. ACE2 is expressed in low numbers of immune cells, higher numbers of HPCs, and up to 65% of rigorously defined HSCs. We also examined effects of exposing HSCs/HPCs and immune cells to SARS-CoV-2 S protein ex vivo. HSCs and HPCs expand less effectively and have less functional colony forming capacity when grown with S protein, while peripheral blood monocytes upregulate CD14 expression and show distinct changes in size and granularity. That these effects are induced by recombinant S protein alone and not the infectious viral particle suggests that simple exposure to SARS-CoV-2 may impact HSCs/HPCs and immune cells via S protein interactions with the cells, regardless of whether they can be infected. These data have implications for immune response to SARS-CoV-2 and for HCT

SETDB1 mediated histone H3 lysine 9 methylation suppresses MLL-fusion target expression and leukemic transformation

Epigenetic regulators play a critical role in normal and malignant hematopoiesis. Deregulation, including epigenetic deregulation, of the HOXA gene cluster drives transformation of about 50% of acute myeloid leukemia. We recently showed that the Histone 3 Lysine 9 methyltransferase SETDB1 negatively regulates the expression of the pro-leukemic genes Hoxa9 and its cofactor Meis1 through deposition of promoter H3K9 trimethylation in MLL-AF9 leukemia cells. Here, we investigated the biological impact of altered SETDB1 expression and changes in H3K9 methylation on acute myeloid leukemia. We demonstrate that SETDB1 expression is correlated to disease status and overall survival in acute myeloid leukemia patients. We recapitulated these findings in mice, where high expression of SETDB1 delayed MLL-AF9 mediated disease progression by promoting differentiation of leukemia cells. We also explored the biological impact of treating normal and malignant hematopoietic cells with an H3K9 methyltransferase inhibitor, UNC0638. While myeloid leukemia cells demonstrate cytotoxicity to UNC0638 treatment, normal bone marrow cells exhibit an expansion of cKit+ hematopoietic stem and progenitor cells. Consistent with these data, we show that bone marrow treated with UNC0638 is more amenable to transformation by MLL-AF9. Next generation sequencing of leukemia cells shows that high expression of SETDB1 induces repressive changes to the promoter epigenome and downregulation of genes linked with acute myeloid leukemia, including Dock1 and the MLL-AF9 target genes Hoxa9, Six1, and others. These data reveal novel targets of SETDB1 in leukemia that point to a role for SETDB1 in negatively regulating pro-leukemic target genes and suppressing acute myeloid leukemia

DEK, a nuclear protein, is chemotactic for hematopoietic stem/progenitor cells acting through CXCR2 and Gαi signaling

Few cytokines/growth modulating proteins are known to be chemoattractants for hematopoietic stem (HSC) and progenitor cells (HPC); stromal cell-derived factor 1α (SDF1α/CXCL12) being the most potent known such protein. DEK, a nuclear DNA-binding chromatin protein with hematopoietic cytokine-like activity, is a chemotactic factor attracting mature immune cells. Transwell migration assays were performed to test whether DEK serves as a chemotactic agent for HSC/HPC. DEK induced dose- and time-dependent directed migration of lineage negative (Lin–) Sca-1+ c-Kit+ (LSK) bone marrow (BM) cells, HSCs and HPCs. Checkerboard assays demonstrated that DEK's activity was chemotactic (directed), not chemokinetic (random migration), in nature. DEK and SDF1α compete for HSC/HPC chemotaxis. Blocking CXCR2 with neutralizing antibodies or inhibiting Gαi protein signaling with Pertussis toxin pretreatment inhibited migration of LSK cells toward DEK. Thus, DEK is a novel and rare chemotactic agent for HSC/HPC acting in a direct or indirect CXCR2 and Gαi protein-coupled signaling-dependent manner

Physioxia enhances T-cell development ex vivo from human hematopoietic stem and progenitor cells

Understanding physiologic T-cell development from hematopoietic stem (HSCs) and progenitor cells (HPCs) is essential for development of improved hematopoietic cell transplantation (HCT) and emerging T-cell therapies. Factors in the thymic niche, including Notch 1 receptor ligand, guide HSCs and HPCs through T-cell development in vitro. We report that physiologically relevant oxygen concentration (5% O2,physioxia), an important environmental thymic factor, promotes differentiation of cord blood CD34+ cells into progenitor T (proT) cells in serum-free and feeder-free culture system. This effect is enhanced by a potent reducing and antioxidant agent, ascorbic acid. Human CD34+ cell-derived proT cells in suspension cultures maturate into CD3+ T cells in an artificial thymic organoid (ATO) culture system more efficiently when maintained under physioxia, compared to ambient air. Low oxygen tension acts as a positive regulator of HSC commitment and HPC differentiation toward proT cells in the feeder-free culture system and for further maturation into T cells in the ATO. Culturing HSCs/HPCs in physioxia is an enhanced method of effective progenitor T and mature T-cell production ex vivo and may be of future use for HCT and T-cell immunotherapies

Mutated Ptpn11 alters leukemic stem cell frequency and reduces the sensitivity of acute myeloid leukemia cells to Mcl1 inhibition

PTPN11 encodes the Shp2 non-receptor protein-tyrosine phosphatase implicated in several signaling pathways. Activating mutations in Shp2 are commonly associated with juvenile myelomonocytic leukemia but are not as well defined in other neoplasms. Here we report that Shp2 mutations occur in human acute myeloid leukemia (AML) at a rate of 6.6% (6/91) in the ECOG E1900 data set. We examined the role of mutated Shp2 in leukemias harboring MLL translocations, which co-occur in human AML. The hyperactive Shp2E76K mutant, commonly observed in leukemia patients, significantly accelerated MLL-AF9-mediated leukemogenesis in vivo. Shp2E76K increased leukemic stem cell frequency and affords MLL-AF9 leukemic cells IL3 cytokine hypersensitivity. As Shp2 is reported to regulate anti-apoptotic genes, we investigated Bcl2, Bcl-xL and Mcl1 expression in MLL-AF9 leukemic cells with and without Shp2E76K. Although the Bcl2 family of genes was upregulated in Shp2E76K cells, Mcl1 showed the highest upregulation in MLL-AF9 cells in response to Shp2E76K. Indeed, expression of Mcl1 in MLL-AF9 cells phenocopies expression of Shp2E76K, suggesting Shp2 mutations cooperate through activation of anti-apoptotic genes. Finally, we show Shp2E76K mutations reduce sensitivity of AML cells to small-molecule-mediated Mcl1 inhibition, suggesting reduced efficacy of drugs targeting MCL1 in patients with hyperactive Shp2

Fate of Hematopoiesis During Aging. What Do We Really Know, and What are its Implications?

This article is made available for unrestricted research re-use and secondary analysis in any form or be any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.There is an ongoing shift in demographics such that older persons will outnumber young persons in the coming years, and with it age-associated tissue attrition and increased diseases and disorders. There has been increased information on the association of the aging process with dysregulation of hematopoietic stem (HSC) and progenitor (HPC) cells, and hematopoiesis. This review provides an extensive up-to date summary on the literature of aged hematopoiesis and HSCs placed in context of potential artifacts of the collection and processing procedure, that may not be totally representative of the status of HSCs in their in vivo bone marrow microenvironment, and what the implications of this are for understanding aged hematopoiesis. This review covers a number of interactive areas, many of which have not been adequately explored. There are still many unknowns and mechanistic insights to be elucidated to better understand effects of aging on the hematopoietic system, efforts that will take multidisciplinary approaches, and that could lead to means to ameliorate at least some of the dysregulation of HSCs and HPCs associated with the aging process.Some of the studies reported in this review, and the writing of this review were supported by the following U.S. Public Health Grants from the National Institutes of Health to H.E.B.: R35 HL139599 (Outstanding Investigator Award), R01 DK109188, U54 DK106846; A.A., J.P.R., and T.T. were supported by T32 DK007519 to H.E.B. R01 HL150624, R56 DK119524, R56 AG052501, DoD W81XWH-13-1-0187, DoD W81XWH-18-1-0265 and DoD W81XWH-19-1-0575, the Leukemia and Lymphoma Society Translational Research Program award 6581-20 and the St. Baldrick’s Foundation Scholar Award to Y.L. MLC was supported by R01 DK109188. CO was supported by National Institute of Allergy and Infectious Diseases (NIAID) contracts HHSN266200500043C and HHSN272201000046C and grants 1U01AI107340-01 and 2R44 AI088288-03A1, National Institute on Aging (NIA) grant R01AG046246-01, and Department of Defense grants PR140896, PR141527, and PR140433P1

Mitigating oxygen stress enhances aged mouse hematopoietic stem cell numbers and function

Bone marrow (BM) hematopoietic stem cells (HSCs) become dysfunctional during aging (i.e., they are increased in number but have an overall reduction in long-term repopulation potential and increased myeloid differentiation) compared with young HSCs, suggesting limited use of old donor BM cells for hematopoietic cell transplantation (HCT). BM cells reside in an in vivo hypoxic environment yet are evaluated after collection and processing in ambient air. We detected an increase in the number of both young and aged mouse BM HSCs collected and processed in 3% O2 compared with the number of young BM HSCs collected and processed in ambient air (~21% O2). Aged BM collected and processed under hypoxic conditions demonstrated enhanced engraftment capability during competitive transplantation analysis and contained more functional HSCs as determined by limiting dilution analysis. Importantly, the myeloid-to-lymphoid differentiation ratio of aged BM collected in 3% O2 was similar to that detected in young BM collected in ambient air or hypoxic conditions, consistent with the increased number of common lymphoid progenitors following collection under hypoxia. Enhanced functional activity and differentiation of old BM collected and processed in hypoxia correlated with reduced “stress” associated with ambient air BM collection and suggests that aged BM may be better and more efficiently used for HCT if collected and processed under hypoxia so that it is never exposed to ambient air O2

Discovery of first-in-class inhibitors of ASH1L histone methyltransferase with anti-leukemic activity

ASH1L histone methyltransferase plays a crucial role in the pathogenesis of different diseases, including acute leukemia. While ASH1L represents an attractive drug target, developing ASH1L inhibitors is challenging, as the catalytic SET domain adapts an inactive conformation with autoinhibitory loop blocking the access to the active site. Here, by applying fragment-based screening followed by medicinal chemistry and a structure-based design, we developed first-in-class small molecule inhibitors of the ASH1L SET domain. The crystal structures of ASH1L-inhibitor complexes reveal compound binding to the autoinhibitory loop region in the SET domain. When tested in MLL leukemia models, our lead compound, AS-99, blocks cell proliferation, induces apoptosis and differentiation, downregulates MLL fusion target genes, and reduces the leukemia burden in vivo. This work validates the ASH1L SET domain as a druggable target and provides a chemical probe to further study the biological functions of ASH1L as well as to develop therapeutic agents

Leukemia Inhibitory Factor Promotes Survival of Hematopoietic Progenitors Ex Vivo and Is Post-Translationally Regulated by DPP4

Hematopoietic cells are regulated in part by extracellular cues from cytokines. Leukemia inhibitory factor (LIF) promotes survival, self-renewal, and pluripotency of mouse embryonic stem cells (mESC). While genetic deletion of LIF affects hematopoietic progenitor cells (HPCs), the direct effect of LIF protein exposure on HPC survival is not known. Furthermore, post-translational modifications (PTM) of LIF and their effects on its function have not been evaluated. We demonstrate that treatment with recombinant LIF preserves mouse and human HPC numbers in stressed conditions when growth factor addition is delayed ex vivo. We show that Lif is upregulated in response to irradiation-induced stress. We reveal novel PTM of LIF where it is cleaved twice by dipeptidyl peptidase 4 (DPP4) protease so that it loses its 4 N-terminal amino acids. This truncation of LIF down-modulates LIF’s ability to preserve functional HPC numbers ex vivo following delayed growth factor addition. DPP4-truncated LIF blocks the ability of full-length LIF to preserve functional HPC numbers. This LIF role and its novel regulation by DPP4 have important implications for normal and stress hematopoiesis, as well as for other cellular contexts in which LIF and DPP4 are implicated