Normal Human Hematopoietic Stem Cells Research Articles

Maintenance of hematopoietic stem cells by non-tyrosine-phosphorylated STAT5 and JAK inhibition

AbstractAdult hematopoietic stem cells (HSCs) are responsible for the lifelong production of blood and immune cells, a process regulated by extracellular cues, including cytokines. Many cytokines signal through the conserved Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway in which tyrosine-phosphorylated STATs (pSTATs) function as transcription factors. STAT5 is a pivotal downstream mediator of several cytokines known to regulate hematopoiesis, but its function in the HSC compartment remains poorly understood. In this study, we show that STAT5-deficient HSCs exhibit an unusual phenotype, including reduced multilineage repopulation and self-renewal, combined with reduced exit from quiescence and increased differentiation. This was driven not only by the loss of canonical pSTAT5 signaling, but also by the loss of distinct transcriptional functions mediated by STAT5 that lack canonical tyrosine phosphorylation (uSTAT5). Consistent with this concept, expression of an unphosphorylatable STAT5 mutant constrained wild-type HSC differentiation, promoted their maintenance, and upregulated transcriptional programs associated with quiescence and stemness. The JAK1/2 inhibitor, ruxolitinib, which increased the uSTAT5:pSTAT5 ratio, had similar effects on murine HSC function; it constrained HSC differentiation and proliferation, promoted HSC maintenance, and upregulated transcriptional programs associated with stemness. Ruxolitinib also enhanced serial replating of normal human hematopoietic stem and progenitor cells (HSPCs), CALR-mutant murine HSCs, and HSPCs obtained from patients with myelofibrosis. Our results therefore reveal a previously unrecognized interplay between pSTAT5 and uSTAT5 in the control of HSC function and highlight JAK inhibition as a potential strategy for enhancing HSC function during ex vivo culture. Increased levels of uSTAT5 may also contribute to the failure of JAK inhibitors to eradicate myeloproliferative neoplasms.

BRD9 regulates normal human hematopoietic stem cell function and lineage differentiation

Bromodomain containing protein 9 (BRD9), a member of the non-canonical BRG1/BRM-associated factor (ncBAF) chromatin remodeling complex, has been implicated as a synthetic lethal target in AML but its function in normal human hematopoiesis is unknown. In hematopoietic stem and progenitor cells (HSPC) genomic or chemical inhibition of BRD9 led to a proliferative disadvantage and loss of stem cells in vitro. Human HSPCs with reduced BRD9 protein levels produced lower numbers of immature mixed multipotent GEMM colonies in semi-solid media. In lineage-promoting culture conditions, cells with reduced BRD9 levels failed to differentiate into the megakaryocytic lineage and showed delayed differentiation into erythroid cells but enhanced terminal myeloid differentiation. HSPCs with BRD9 knock down (KD) had reduced long-term multilineage engraftment in a xenotransplantation assay. An increased number of downregulated genes in RNAseq analysis after BRD9 KD coupled with a gain in chromatin accessibility at the promoters of several repressive transcription factors (TF) suggest that BRD9 functions in the maintenance of active transcription during HSC differentiation. In particular, the hematopoietic master regulator GATA1 was identified as one of the core TFs regulating the gene networks modulated by BRD9 loss in HSPCs. BRD9 inhibition reduced a GATA1-luciferase reporter signal, further suggesting a role for BRD9 in regulating GATA1 activity. BRD9 is therefore an additional example of epigenetic regulation of human hematopoiesis.

Simultaneous inhibition of Sirtuin 3 and cholesterol homeostasis targets acute myeloid leukemia stem cells by perturbing fatty acid β-oxidation and inducing lipotoxicity.

Outcomes for patients with acute myeloid leukemia (AML) remain poor due to the inability of current therapeutic regimens to fully eradicate disease initiating leukemia stem cells (LSCs). Previous studies have demonstrated that oxidative phosphorylation (OXPHOS) is an essential process that is targetable in LSCs. Sirtuin 3 (SIRT3), a mitochondrial deacetylase with a multi-faceted role in metabolic regulation, has been shown to regulate OXPHOS in cancer models; however, it has not yet been studied in the context of LSCs. Thus, we sought to identify if SIRT3 is important for LSC function. Using RNAi and a SIRT3 inhibitor (YC8-02), we demonstrate that SIRT3 is a critical target for the survival of primary human LSCs but is not essential for normal human hematopoietic stem and progenitor cell (HSPC) function. To elucidate the molecular mechanisms by which SIRT3 is essential in LSCs we combined transcriptomic, proteomic, and lipidomic approaches, showing that SIRT3 is important for LSC function through the regulation of fatty acid oxidation (FAO) which is required to support oxidative phosphorylation and ATP production in human LSCs. Further, we discovered two approaches to further sensitize LSCs to SIRT3 inhibition. First, we found that LSCs tolerate the toxic effects of fatty acid accumulation induced by SIRT3 inhibition by upregulating cholesterol esterification. Disruption of cholesterol homeostasis sensitizes LSCs to YC8-02 and potentiates LSC cell death. Second, SIRT3 inhibition sensitizes LSCs to BCL-2 inhibitor venetoclax. Together, these findings establish SIRT3 as a regulator of lipid metabolism and potential therapeutic target in primitive AML cells.

Modeling Premalignant Transformation of Hematopoietic Stem Cells in a Nanobioreactor in Microgravity

Microgravity coupled with increased radiation exposure aboard the ISS provides a unique environment to simulate and study response to injury, inflammatory signaling, aging, and (pre-)malignant transformation of normal human hematopoietic stem cells (HSCs) in an accelerated timeframe. The NASA Twin study suggests that genomic, epigenomic, epitranscriptomic, and proteomic changes may detrimentally impact hematopoietic stem and immune cell fitness and induce stem cell exhaustion (Garrett-Bakelman et al., Science, 2019). Moreover, changes indicative of pre-cancer stem cell generation, such as increased chromosome translocations and inversions, occurred and persisted post-flight. Additionally, a recent publication entitled Multisystem Toxicity in Cancer: Lessons from NASA's Countermeasures Program found significant similarities between the multisystem physiological toxicities experienced in cancer patients as well as during spaceflight (Scott et al, Cell 2019). These reports highlight the benefits of studying injury response, changes in mutational profiles and cancer evolution in microgravity at the stem cell level. For this study, we designed a novel bioreactor system to support the culture of donor-derived human HSCs in low Earth orbit (LEO). A sponge matrix and stromal cells model the microenvironment HSCs reside in within the bone marrow niche. Testing on Earth confirmed our system's ability to maintain stem cell fitness over several weeks. To assess stem cell physiology in LEO over time, we lentivirally transduced a reporter into the HSCs pre-flight (Pineda et al., Scientific Reports 2016), which allows for cell cycle tracking via fluorescence imaging. This will provide data for assessment of stem cell health, maintenance and functionality. Furthermore, we are analyzing cytokine profiles and mutational status post-flight, with a focus on signatures we have previously connected to (pre-)malignant transformation via RNA sequencing analysis (Jiang, Cancer Cell 2019). The first batch of bioreactors launched with the SpX-24 mission in December 2021 and the second batch is currently on orbit as part of the SpX-25 mission. This investigation has the potential to provide valuable insights into the maintenance of hematopoietic stem cell health and functionality, HSC response to injury through accumulation of mutations and, eventually, the mechanisms fueling long-term (pre-)malignant transformation into leukemia stem cells.

PRL2 phosphatase enhances oncogenic FLT3 signaling via dephosphorylation of the E3 ubiquitin ligase CBL at tyrosine 371

AbstractAcute myeloid leukemia (AML) is an aggressive blood cancer with poor prognosis. FMS-like tyrosine kinase receptor-3 (FLT3) is one of the major oncogenic receptor tyrosine kinases aberrantly activated in AML. Although protein tyrosine phosphatase PRL2 is highly expressed in some subtypes of AML compared with normal human hematopoietic stem and progenitor cells, the mechanisms by which PRL2 promotes leukemogenesis are largely unknown. We discovered that genetic and pharmacological inhibition of PRL2 significantly reduce the burden of FLT3-internal tandem duplications–driven leukemia and extend the survival of leukemic mice. Furthermore, we found that PRL2 enhances oncogenic FLT3 signaling in leukemia cells, promoting their proliferation and survival. Mechanistically, PRL2 dephosphorylates the E3 ubiquitin ligase CBL at tyrosine 371 and attenuates CBL-mediated ubiquitination and degradation of FLT3, leading to enhanced FLT3 signaling in leukemia cells. Thus, our study reveals that PRL2 enhances oncogenic FLT3 signaling in leukemia cells through dephosphorylation of CBL and will likely establish PRL2 as a novel druggable target for AML.

Abstract 3154: Assessing hematopoietic stem cell fitness within a nanobioreactor in microgravity

Abstract Microgravity coupled with increased radiation exposure aboard the ISS provides a unique environment to simulate and study response to injury, inflammatory signaling, aging, and (pre-)malignant transformation of normal human hematopoietic stem cells (HSCs) in an accelerated timeframe. The NASA Twins study suggests that genomic, epigenomic, epitranscriptomic, and proteomic changes may detrimentally impact hematopoietic stem and immune cell fitness and induce stem cell exhaustion (Garrett-Bakelman et al., Science, 2019). Moreover, changes indicative of pre-cancer stem cell generation such as increased chromosome translocations and inversions occurred and persisted post-flight. Additionally, a recent publication entitled Multisystem Toxicity in Cancer: Lessons from NASA’s Countermeasures Program found significant similarities between the multisystem physiological toxicities in cancer patients and during spaceflight (Scott et al, Cell 2019). These reports highlight the benefits of studying injury response, changes in mutational profiles and cancer evolution in microgravity at the stem cell level. For this study, we designed a novel bioreactor system to support the culture of donor-derived human HSCs in low Earth orbit (LEO). A sponge matrix and stromal cells model the microenvironment HSCs reside in within the bone marrow niche. Testing on Earth confirmed our system’s ability to maintain stem cell fitness over several weeks. To assess stem cell physiology in LEO over time, we lentivirally transduce a reporter into the HSCs pre-flight (Pineda et al., Scientific Reports 2016), which allows for cell cycle tracking via fluorescence imaging. This will provide data for assessment of stem cell health, maintenance and functionality. Furthermore, we will be analyzing mutational status post-flight, with a focus on signatures we have previously connected to (pre-)malignant transformation via RNA sequencing analysis (Jiang, Cancer Cell 2019). Our bioreactors are scheduled to launch as part of the SpaceX CRS-24 mission on Dec 21, 2021. This investigation may provide valuable insights into the maintenance of hematopoietic stem cell health and functionality, response to injury through accumulation of mutations and, eventually, the mechanisms fueling long-term (pre-)malignant transformation into leukemia stem cells. Citation Format: Luisa Ladel, Jessica Pham, Isabelle Oliver, Larisa Balaian, Catriona HM Jamieson. Assessing hematopoietic stem cell fitness within a nanobioreactor in microgravity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3154.

Immunotherapy Targeting ST2/IL-33 Signaling in Myeloid Leukemia Stem Cells

▪Therapies for acute myeloid leukemia (AML) has barely changed over 30 years, while treatment for other blood cancers have made remarkable leaps forward. Recent advances in genomics have allowed molecular targeted therapies (i.e. FLT3-ITD, IDH, c-KIT inhibitors) extending survival, but most patients still succumb (Burd et al. Nat Med 2020). Therefore, developing more efficient, less toxic, and immune-based therapies for AML is an urgent unmet need. Previous studies showed that stromal cell-derived IL-33 stimulates myeloproliferative neoplasms (Mager et al. J Clin Invest 2015). Stimulation-2 (ST2), IL-33 receptor, contributes to leukemia stem cells (LSCs) survival in Cbfb-MYH11 mice (Wang et al . Sci Rep 2019). We showed that ST2 blockade enhanced graft versus leukemia activity against MLL-AF9 egfp AML after hematopoietic cell transplantation (Zhang et al . Sci Transl Med 2015). We and others, also, found that ST2 is expressed on normal murine and human hematopoietic stem cells (HSCs), respectively (Capitano et al. Blood Cells Mol Dis 2020; Alt et al. Biol Blood Marrow Transplant 2019). These data suggest a leukemia-promoting role of ST2/IL-33 signaling.To determine clinical relevance of ST2 in AML, we generated Kaplan-Meier curves using TCGA (n=173) and TARGET AML (n=187) databases. Decreased survival was observed in patients with high IL1RL1 (ST2 gene) which was validated in an independent database (AMLCG 1999 trial, n=417) (Fig. 1A). Since ST2 is expressed on HSCs, we interrogated if ST2 is expressed on LSCs defined as CD34 +CD38 - in the Princess Margaret Leukemia biobank (n=192), and found ST2 is higher on LSCs vs CD34 -CD38 +/- cells (Fig. 1B). We then sought to analyze ST2 on bone marrow samples comparing complete responders vs refractory patients to note that ST2 expression was increased in refractory patients' LSCs (Fig. 1C). To scrutinize the role of ST2 in initiating leukemogenesis, we performed limiting dilution transplantation using 500, 200, and 50 Lin -Sca-1 +c-KIT +-sorted LSCs from WT vs ST2 -/- MLL-AF9 egfp transduced cells. Frequency of LSCs in ST2 -/- cells was decreased by ~15-fold as opposed to WT cells [1:2141 (1:546-1:8405) vs 1:145 (1:75-1:283), p=3.37e-05] (Fig. 2A). We also tested leukemia maintenance, secondary transplantations from the primary recipients resulted in leukemia growth delay in ST2 -/- vs WT cells which was confirmed in tertiary transplantations (Fig. 2B-E). Self-renewal ability of LSCs is correlated to reactive oxygen species (ROS) (Testa et al. Exp Hematol 2016), and we found that ROS levels in ST2 -/- leukemic cells are markedly diminished in contrast to WT leukemic cells (Fig. 2F). ST2 deficiency in leukemic cells arrests G2/S/M cell cycle progression in LSCs (Fig. 2G-J). These data indicated that ST2 is indispensable for initiating and maintaining LSCs in MLL-AF9 AML.We next developed murine and human Fc-silenced-bispecific antibodies engaging mouse or human ST2 and CD3 (BsAb) built on the IgG[L]-scFv platform with proven ability to drive T cells into human tumors for effective tumor ablation (Santich et al. Sci Transl Med 2020; Park et al. J Immunother Cancer 2021) (Fig. 3A, 3E). Both BsAbs showed >90% purity by HPLC, stability under heat stress and low endotoxin. Animals did not exhibit any in vivo toxicity at BsAb doses of 0.4, 2, 5, 10 μg ip q 3 days x 6 doses (not shown). In the immunocompetent MLL-AF9 mice, murine anti-ST2 BsAb (BC281) treatment (10 μg i.p, 4 days post-AML challenge and given every three days for a total of 6 injections) resulted in extended survival compared to isotype control mice (Fig. 3B). Leukemic cells and LSCs were accordingly decreased in treated vs control group (Fig. 3C-D). We modeled humanized leukemic mice with MOLM-14 egfp cells and weekly injection of human CD8 + T cells in NOD.Cg-Prkdc scid Il2rg tm1Wjl/SzJ (NSG) mice (Fig. 3F). Animals treated with human anti-ST2 BsAb (BC282), using a similar regimen as for the immunocompetent model, led to better survival when compared to animals treated with mutated non-functional anti-ST2 BsAb (BC283) (Fig. 3G). Frequency of MOLM-14 egfp cells was lower in the BC282 vs BC283 group (Fig. 3H). These results suggested that anti-ST2 BsAbs can inhibit AML growth to improve survival.We concluded that ST2 is a potential therapeutic target, and ST2-specific T cell engaging BsAbs represent promising immunotherapeutics for AML. [Display omitted] DisclosuresCheung: Medical University of South Carolina: Patents & Royalties: inventor on the ST2 bispecific antibody patent application; Y-mabs Therapeutics and Abpro-Labs Inc: Patents & Royalties: inventor on multiple patents filed by MSK, including those licensed to Ymabs Therapeutics, Biotec Pharmacon, and Abpro-labs; Eureka Therapeutics: Membership on an entity's Board of Directors or advisory committees. Paczesny: Medical University of South Carolina: Patents & Royalties: inventor on the ST2 bispecific antibody patent application.

Abstract 3099: The metabolic enzyme hexokinase 2 localizes to the nucleus in AML and normal hematopoietic stem/progenitor cells to maintain stemness

Abstract Mitochondrial metabolites affect epigenetic marks, but it is largely unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stem cell function in AML. Here, we discovered that the mitochondrial enzyme, Hexokinase 2 (HK2), localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stem cell function. We searched for mitochondrial enzymes moonlighting in the nucleus using 8227 AML cells, a primary AML culture model arranged in a hierarchy with defined stem cells. By immunoblotting and confocal microscopy, we detected HK2 in the nucleus of 8227 cells with higher expression in the nucleus of stem cells vs bulk cells. In contrast, other metabolic enzymes including PFK1, FH, PKM2, GPI1, ENO1, CS, ACO2, and SDHA1, 2 were not detected in the nucleus of these cells. We also detected HK2 in the nucleus of AML cell lines as well as 7 of 9 primary AML samples. Next, we tested whether nuclear HK2 was functionally important to maintain stem cell function in AML. We over-expressed HK2 tagged with nuclear localizing signals in 8227 and NB4 leukemia cells. This increased clonogenic growth and inhibited retinoic acid-mediated cell differentiation without changing basal proliferation. Nuclear HK2 also increased engraftment of 8227 cells into mouse marrow. We evaluated the selective inhibition of nuclear HK2 by over-expressing HK2 with an outer mitochondrial localization signal while knocking down endogenous HK2 with 3'UTR shRNA. Selective depletion of nuclear HK2 reduced clonogenic growth, increased AML differentiation with ATRA, and decreased CD34+CD38- 8227 stem cells without changing basal proliferation. Nuclear HK2 was also higher in normal human hematopoietic stem cells and multipotent progenitor fractions and declined as the cells matured. Over-expression of nuclear HK2 in normal cord blood increased the primary and secondary engraftment into mice. Transgenic mice over-expressing nuclear HK2 driven by a Vav promoter had increased hematopoietic stem cells in the marrow and decreased monocytes and lymphocytes in the peripheral blood. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2 (D209A D657A) and observed increased clonogenic growth and inhibited differentiation on ATRA treatment, nuclear HK2 function is independent of its kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry identified proteins related to chromatin organization and regulation to interact with nuclear HK2. In summary, we discovered that HK2 localizes to nucleus of AML cells and functions independent of its kinase activity to maintain the stem/progenitor state of AML. Thus, we define a new role for mitochondrial enzymes in the regulation of leukemic stemness and differentiation. Citation Format: Geethu Emily Thomas, Grace Egan, Laura Gracia Prat, Aaron Botham, Veronique Voisin, Elias Orouji, Jordan Chin, Boaz Nachmias, Kerstin B. Kaufmann, Neil Maclean, Rose Hurren, Marcela Gronda, Xiaoming Wang, Dilshad H. Khan, Rashim P. Singh, Andrea Arruda, Mark Minden, Gary D. Bader, John E. Dick, Aaron D. Schimmer. The metabolic enzyme hexokinase 2 localizes to the nucleus in AML and normal hematopoietic stem/progenitor cells to maintain stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3099.

Identification of Novel Targets for Antibody or Chimeric Antigen Receptor (CAR) Based Immunotherapy in AML: Characterizing the Surfaceome from Primary Patient Samples

Background: Despite considerable advances in the field of immunotherapy of acute myeloid leukemia, on-target off-leukemia toxicity remains a challenge. Thus, novel target antigens are sought after. The Cell Surface Capture (CSC) technology enables a discovery-driven approach to detect surface proteins. However, direct processing of primary samples has been limited by the substantial number of viable cells needed for the CSC-workflow. We therefore addressed this issue by improving the technique including the use of our ex vivo culture system. This allowed for an unbiased, direct assessment of primary AML patient samples and thus enabled us to interrogate the AML surfaceome in clinically relevant samples.Methods: Primary cells from patients with newly diagnosed or relapsed AML were cultured in our ex vivo co-culture system as previously described (Krupka et al. 2014) using MS-5 feeder cells. After 3 days, non-adherent cells were harvested and immediately subjected to the CSC-workflow. Glyco-CSC and its variants Cys-Glyco-CSC and Lys-CSC were initially performed as described by Wollscheid and colleagues (Wollscheid et al. 2009 and Bausch-Fluck et al. 2012) and subsequently modified to improve the yield on AML samples. CSC samples were analyzed by tandem mass spectrometry on an Orbitrap Elite instrument and modified peptides were identified using MaxQuant software.Results: To enhance the number of successfully identified surface proteins, we adapted the original protocol. These modifications doubled the yield in identified proteins from AML cell lines from initially 125 to 252 in the modified protocol. More importantly, the modifications increased the specificity of the assay significantly. In the original Glyco-CSC experiments, only 54% of all identified peptides displayed a mass shift (of 0.984 Da) associated with successful N-glycosylation and had a transmembrane domain or a signal peptide annotated in UniProt. After modification of the protocol, 80.4% of all peptides fulfilled these criteria. The modified protocol was therefore used for all primary samples. 5 representative primary patient samples from initial diagnosis and 2 samples from relapsed disease were analyzed. All samples yielded sufficient viable cell numbers after ex vivo culture and could successfully be subjected to the CSC workflow. We identified a total of 719 surface proteins fulfilling all filter rules. 22.9% of these proteins had CD annotations. Next, we only considered proteins that were detected by CSC in at least half of the primary patient samples tested. In addition, proteins were filtered to eliminate targets that are abundantly expressed on normal human hematopoietic stem and progenitor cells as well as relevant healthy tissue using publicly available transcriptome databases. 84 proteins were selected as potential candidates for manual screening. Of note, the expression of several antigens currently under investigation for AML immunotherapy (i.e. CD33, CD123, CD135, CLL-1) were detected by the method. We selected 6 promising novel candidate markers previously not described as relevant targets in AML. These were assessed by FACS analysis in independent patient samples. 3/6 of our novel targets showed uniform expression in all independent primary AML samples tested (defined as MFI ratio >1.5).Conclusion: In conclusion, improvements in the CSC-Workflow combined with our ex vivo culture system allowed for the successful identification of the AML surfaceome from primary patient samples without the necessity of xeno-amplification. We identified 6 novel targets, 3/6 were found to be uniformly expressed in independent primary AML samples. These candidates are now being evaluated further as potential targets for antibody and CAR based immunotherapy in AML. DisclosuresNo relevant conflicts of interest to declare.

Selective antisense oligonucleotide inhibition of human IRF4 prevents malignant myeloma regeneration via cell cycle disruption.

In multiple myeloma, inflammatory and anti-viral pathways promote disease progression and cancer stem cell generation. Using diverse pre-clinical models, we investigated the role of interferon regulatory factor 4 (IRF4) in myeloma progenitor regeneration. In a patient-derived xenograft model that recapitulates IRF4 pathway activation in human myeloma, we test the effects of IRF4 antisense oligonucleotides (ASOs) and identify a lead agent for clinical development (ION251). IRF4 overexpression expands myeloma progenitors, while IRF4 ASOs impair myeloma cell survival and reduce IRF4 and c-MYC expression. IRF4 ASO monotherapy impedes tumor formation and myeloma dissemination in xenograft models, improving animal survival. Moreover, IRF4 ASOs eradicate myeloma progenitors and malignant plasma cells while sparing normal human hematopoietic stem cell development. Mechanistically, IRF4 inhibition disrupts cell cycle progression, downregulates stem cell and cell adhesion transcript expression, and promotes sensitivity to myeloma drugs. These findings will enable rapid clinical development of selective IRF4 inhibitors to prevent myeloma progenitor-driven relapse.

Chronic lymphocytic leukemia B-cell-derived TNFα impairs bone marrow myelopoiesis.

SummaryTNFα is implicated in chronic lymphocytic leukemia (CLL) immunosuppression and disease progression. TNFα is constitutively produced by CLL B cells and is a negative regulator of bone marrow (BM) myelopoiesis. Here, we show that co-culture of CLL B cells with purified normal human hematopoietic stem and progenitor cells (HSPCs) directly altered protein levels of the myeloid and erythroid cell fate determinants PU.1 and GATA-2 at the single-cell level within transitional HSPC subsets, mimicking ex vivo expression patterns. Physical separation of CLL cells from control HSPCs or neutralizing TNFα abrogated upregulation of PU.1, yet restoration of GATA-2 required TNFα neutralization, suggesting both cell contact and soluble-factor-mediated regulation. We further show that CLL patient BM myeloid progenitors are diminished in frequency and function, an effect recapitulated by chronic exposure of control HSPCs to low-dose TNFα. These findings implicate CLL B-cell-derived TNFα in impaired BM myelopoiesis.

3D Genomics of Acute Meyloid Leukemia Reveals the Imbalance between DNA Methylation Canyon Interactions and Leukemic Specific Enhancer Network Interactions

Changes in 3D chromatin organization like enhancer hijacking are believed to the driver for disease development like leukemia. Here we performed high-resolution HiC assays on primary acute myeloid leukemia (AML) samples and cell lines to dissect the abnormal 3D chromatin organization in AML. Our data set covers 5 AML samples and 3 AML cell lines. This dataset includes the common genetic abnormalities in AML: MLL-rearrangement, NPM1 mutation, RUNX1 mutation, and IDH1/TET2 mutations. We have recently generated high-resolution map for normal human hematopoietic stem cells (HSC) (Zhang et al. Mole Cell. 2020). In comparison with the HSC 3D chromatin organization, we found TADs and loops are very stable in both primary leukemia samples and cell lines. Less than 5% of all TADs in HSC fuse in AML, mimicking the enhancer hijacking scenario. These fusion events do not cause the gene expression changes of genes in the fused TAD. Interestingly, in TET2 or IDH1 mutated AML blast, two-fold more TAD fusion events occurred in primary AML blast in comparison with RUNX1 and MLL-r leukemia, with a loss in the CTCF sites on the TAD fusion break point. We previously found in HSC, the Polycomb marked DNA methylation Canyons (DMC) form multi-Mb size long-range interactions. DMC interactions in general decrease in primary AMLs. AMLs with IDH1 or TET2 mutations shows the biggest reduction in DMC interactions. Hypermethylation in the DMCs is observed in the AML samples with IDH1/2 or TET2 mutations, suggesting DNA methylation level in DMCs controls DMC 3D interactions directly. In leukemia cell lines, the DMC interactions almost disappear, with further hypermethylation in DMCs. Compared with normal HSC, we found in AML, the AML-specific H3K27ac marked regions form leukemia specific loops and transcription stripes in both cell lines and primary samples. Particularly in MLL-r primary leukemias, we found broad H3K27ac covered, hyperacetylated domains (10kb to 200kb). 22 such hyperacetylated domains were identified and associated with leukemogenic genes such as SATB1, ZEB2 and HOXA. All these domains formed distinct 3D micro TAD in the MLL-r primary leukemia in comparison with the HSPC, and CTCFs are not located at the border of these domains. Taken together, suggest active leukemia specific transcription created new 3D genomic interactions which is independent of cohesion-CTCF mediated loop extrusion. Interestingly, in HOXA cluster, we found a geneless DMC 1.3MB upstream of HOXA switched from Polycomb binding site to active enhancer site in the leukemia cells. By applying CRISPR/Cas9 editing, we found this canyon is essential for survival of HOXA high expressing leukemia cell lines like OCI-AML3 and MV4:11. In summary, we found the 3D chromatin organization in human leukemia significantly alters in two opposite way 1. The significant loss of Polycomb marked DMC interactions caused by the DNA hypermethylation and 2. The leukemic specific hyperacetylated domains form its own distinct micro TAD and stripes in the 3D chromatin organization. Disclosures No relevant conflicts of interest to declare.

Selective Antisense Oligonucleotide Inhibition of Human IRF4 Prevents Malignant Myeloma Regeneration Via Cell Cycle Disruption

In multiple myeloma, pro-inflammatory factors such as interleukin-6 signaling and downstream anti-viral response pathway activation have been correlated with disease progression, symptom severity, and cancer stem cell generation. However, the role of interferon-response factors in the maintenance of malignant progenitors has not been explored. We hypothesized that the key B-cell progenitor fate determinant, interferon-response factor-4 (IRF4), governs regeneration of drug-resistant malignant progenitors in multiple myeloma. Using diverse pre-clinical models, including a humanized in vivo model that recapitulates IRF4 pathway activation detected by RNA-sequencing in primary high-risk samples, we tested whether direct targeting of human IRF4 using state-of-the-art antisense oligonucleotide (ASO) agents could impair myeloma regeneration. While human IRF4 overexpression expanded a putative progenitor population in vitro, IRF4 ASO treatment impaired myeloma cell survival commensurate with reduced IRF4 and MYC gene and protein expression. In addition to impeding subcutaneous tumor formation and myeloma dissemination in xenograft immunocompromised mouse models, human IRF4-targeted ASO monotherapy significantly improved overall animal survival. Moreover, in a patient-derived mouse model of high-risk myeloma, treatment with a lead IRF4 ASO that will advance to a Phase 1 trial (NCT04398485) eradicated CD138-negative MM progenitors and their malignant plasma cell progeny at doses that spared normal human hematopoietic stem cells and B cell development. Complementary mechanistic studies uncovered a role for IRF4 inhibition in disrupting microenvironment-responsive cell cycle progression, down-regulating stem cell regulatory and cell adhesion transcript expression including MYC and CXCR4, and promoting sensitivity to standard-of-care therapeutic agents. These comprehensive therapeutic index and proof-of-concept studies will enable rapid clinical development of IRF4 inhibition to prevent myeloma relapse driven by retention of dormant, drug-resistant progenitors in inflammatory microenvironments.

Differences in expression and function of LEF1 isoforms in normal versus leukemic hematopoiesis.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults and is propagated by leukemic stem cells (LSCs), often characterized by deregulated Wnt signaling. We previously showed that the central transcriptional mediator of Wnt signaling LEF1 is able to cause AML in mice and acts as an independent prognostic factor in normal karyotype AML. Here, we show that treatment naïve normal karyotype AML as well as samples AML LSCs predominantly express the long β-catenin-binding isoform of LEF1 in sharp contrast to normal human hematopoietic stem cells, which lack expression of the long isoform, but express the short N-terminally truncated isoform with loss of the β-catenin-binding site. Gene expression and ChiP-Seq analyses in mice linked the long isoform to Wnt-β-catenin signaling and oncogenic pathways, the N-terminally truncated isoform to stemness associated genes. Approaches impairing binding of LEF1 to β-catenin significantly impaired AML growth, but spared normal hematopoietic stem cells. This report now demonstrates a striking difference of LEF1 isoform expression between normal and AML cells, contributing to higher vulnerability of leukemic cells to approaches targeting β-catenin/LEF1 interaction.

Anti-CD117 antibody depletes normal and myelodysplastic syndrome human hematopoietic stem cells in xenografted mice

AbstractThe myelodysplastic syndromes (MDS) represent a group of clonal disorders that result in ineffective hematopoiesis and are associated with an increased risk of transformation into acute leukemia. MDS arises from hematopoietic stem cells (HSCs); therefore, successful elimination of MDS HSCs is an important part of any curative therapy. However, current treatment options, including allogeneic hematopoietic cell transplantation (HCT), often fail to ablate disease-initiating MDS HSCs, and thus have low curative potential and high relapse rates. Here, we demonstrate that human HSCs can be targeted and eliminated by monoclonal antibodies (mAbs) that bind cell-surface CD117 (c-Kit). We show that an anti-human CD117 mAb, SR-1, inhibits normal cord blood and bone marrow HSCs in vitro. Furthermore, SR-1 and clinical-grade humanized anti-human CD117 mAb, AMG 191, deplete normal and MDS HSCs in vivo in xenograft mouse models. Anti-CD117 mAbs also facilitate the engraftment of normal donor human HSCs in MDS xenograft mouse models, restoring normal human hematopoiesis and eradicating aggressive pathologic MDS cells. This study is the first to demonstrate that anti-human CD117 mAbs have potential as novel therapeutics to eradicate MDS HSCs and augment the curative effect of allogeneic HCT for this disease. Moreover, we establish the foundation for use of these antibody agents not only in the treatment of MDS but also for the multitude of other HSC-driven blood and immune disorders for which transplant can be disease-altering.

Abstract SY39-03: Stem cell competition is central to leukemogenesis

Abstract Tissue stem cells regenerate the body and are the only cells that maintain self-renewal throughout life. When stem cells divide they give rise to stem cells (by self-renewal) and progenitors (by differentiation). Stem cell isolation and transplantation is the basis for regenerative medicine. We isolated mouse and then human hematopoietic stem cells (HSCs). Importantly, we demonstrated that transplantation of purified HSCs results in complete regeneration of the blood and immune systems without causing graft vs. host disease, and can induce permanent transplant tolerance of any organ or cell from the HSC donor. In a clinical trial in metastatic breast cancer patients, HSC purification made autologous transplantation to cancer patients possible, without including cancerous cells in the graft. The 15-year overall survival rate was ~7% in recipients of mobilized peripheral blood (MPB) autologous transplantation, and ~33% in recipients of cancer-free HSCs. We have also prospectively isolated human fetal brain CNS stem cells and found a method to propagate them without changing phenotype or normal regenerative functions upon transplantation. These are and have been used in clinical trials with children with genetic neurodegenerative diseases such as Batten and Pelizeus Mehrbacher, in patients with thoracic and cervical spinal cord injuries, and in patients with age-related macular degeneration. Self-renewal is strictly regulated and restricted to stem cells, because deregulation can lead to cancer. To study the relationship between stem cells and cancer, we followed the progression from hematopoietic stem cells (HSCs) to myelogenous leukemias. We found that the developing precancerous clones progress and accumulate mutations at the stage of HSCs, until they become fully malignant. At this point, the “leukemia” stem cell is a downstream oligolineage or multilineage progenitor that has evaded programmed cell death and programmed cell removal, and also acquired self-renewal. In the case of chronic myeloid leukemia (CML), bcr-abl+ HSC clones outcompete normal HSCs in the chronic phase. The transition from CML to myeloid blast crisis results in the leukemia stem cells appearing in the granulocyte-macrophage progenitor (GMP) stage, and is accompanied by cell intrinsic activation of β-catenin, endowing the already mutant progenitors with a self-renewal capacity. While there are many ways to defeat programmed cell death and senescence, there appears to be one dominant method that cancer cells use to avoid programmed cell removal—the elevated expression of the cell surface “don't eat me” protein CD47, the ligand for macrophage SIRPα. All cancers tested express CD47 to overcome expression of “eat me” signals such as calreticulin that is produced by macrophages and adheres to asialogylycoproteins on the surface of cancer cells. Antibodies that block the CD47-SIRPα interaction enable phagocytosis and killing of the tumor cells in vitro and in vivo. This includes human solid tumors and lymphomas/leukemias/myelomas. We showed that anti-CD47 antibodies or high-affinity SIRPα proteins synergize with anti-CD20 antibodies to eliminate human non-Hodgkin lymphoma in immune-deficient mice. We generated a humanized antibody of the IgG4 isotype, and these compounds are currently being tested in clinical trials. Citation Format: Irving L. Weissman. Stem cell competition is central to leukemogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY39-03.

Chromosome positioning in interphase nuclei of hematopoietic stem cell and myeloid precursor.

Human myelopoiesis is an intriguing biological process during which multipotent stem cells limit their differentiation potential generating precursors that evolve into terminally differentiated cells. The differentiation process is correlated with differential gene expression and changes in nuclear architecture. In interphase, chromosomes are distinct entities known as chromosome territories and they show a radial localization that could result in a constrain of inter-homologous distance. This element plays a role in genome stability and gene expression. Here, we provide the first experimental evidence of 3D chromosomal arrangement considering two steps of human normal myelopoiesis. Specifically, multicolor 3D-FISH and 3D image analysis revealed that, in both normal human hematopoietic stem cells and myelod precursors CD14-, chromosomal position is correlated with gene density. However, we observed that inter-homologue distances are totally different during differentiation. This could be associated with differential gene expression that we found comparing the two cell types. Our results disclose an unprecedented framework relevant for deciphering the genomic mechanisms at the base of normal human myelopoiesis.

Abstract 39: XPO1 inhibitor, KPT-8602, is well tolerated and highly active against AML blasts and LICs

Abstract Standard chemotherapy often induces remissions in AML patients, but the disease frequently relapses due to incomplete targeting of leukemia-initiating cells (LICs), emphasizing the need for novel effective treatments. An attractive target for AML therapy is nuclear exporter exportin 1 (XPO1), which mediates continuous nuclear export of a subset of proteins and RNAs. The orally bioavailable XPO1 inhibitor of the Selective Inhibitor of Nuclear Export (SINE) class of compounds, selinexor (KPT-330), is currently in phase 1 and 2 clinical trials in adult patients with AML and in phase 1 trials for relapsed childhood ALL and AML. The results of these trials are encouraging, as they have demonstrated that selinexor is active in inducing remission in patients with relapsed or refractory disease. Here we report the antileukemic efficacy and tolerability of KPT-8602, a second-generation XPO1 inhibitor. KPT-8602 demonstrates substantially reduced brain penetration compared to selinexor, with resultant attenuation of the CNS-mediated side effects of anorexia and weight loss. Due to its improved tolerability profile, KPT-8602 can be given daily compared to the two- or three-times-weekly regimen of selinexor. To define the antileukemia activity of KPT-8602 against primary AML blasts and LICs in a relevant preclinical setting, we established patient-derived xenograft (PDX) models, in which leukemic blasts from AML patients with poor-prognosis disease (cytogenetically normal AML with FLT3-ITD (AML-CN), AML with complex karyotype (AML-CK), and MDS-derived AML (MDS/AML) are transplanted into immunodeficient NOD-SCID-IL2Rcgnull (NSG) mice. Mice engrafted with leukemic blasts are treated with vehicle, selinexor (20 mg/kg three times per week), or KPT-8602 (15 mg/kg daily for 4 weeks). KPT-8602 exhibits superior antileukemic activity and better tolerability in the AML PDX models tested, with nearly complete elimination of human AML cells in AML-CN model. To determine the frequency of LICs in the vehicle control, selinexor or KPT-8602, we used a limiting dilution transplantation assay. Although both selinexor and KPT-8602 effectively targeted LICs, KPT-8602 is more potent in eliminating LICs, as evidenced by a more profound reduction of LIC frequency in the AML-CK and AML-CN PDX models (507-fold vs. 0.9-fold reduction in AML-CK, P<0.0001; and 437-fold vs. 111-fold reduction in AML-CN by KPT-8602 vs. selinexor, P=0.79). The impact on normal human hematopoietic stem and progenitor cell (HSPC) fractions by CD34/CD38 flow analysis and limiting dilution transplantation assays (performed by the same methodology as described for PDX models of AML) demonstrates that, in contrast to its profound effects against AML LIC, KPT-8602 does not preferentially kill normal HSPC, providing a therapeutic window for elimination of relapse-driving LICs while sparing normal HSPCs. KPT-8602 has entered a phase 1/2 trial in patients with relapsed/refractory multiple myeloma. Our preclinical results indicate that KPT-8602 should be tested in clinical trials to determine its toxicity and efficacy in patients with relapsed or refractory AML, with the hope that it can eventually be integrated into upfront combination chemotherapy regimens. To delineate the pathways that mediate the apoptotic signaling in response to the SINE compounds, we are conducting the RNA-seq, ChIP-seq and ATAC-seq studies on AML cell lines treated in vitro as well as AML cells isolated from PDX mice. The preliminary results of these studies will be presented at the meeting. Citation Format: Julia Etchin, Alla Berezovskaya, Amy S. Conway, Ilene A. Galinsky, Richard M. Stone, Erkan Baloglu, William Senapedis, Yosef Landesman, Michael Kauffman, Sharon Shacham, Jean CY Wang, A Thomas Look. XPO1 inhibitor, KPT-8602, is well tolerated and highly active against AML blasts and LICs [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 39.

Mutant nucleophosmin1 (NPM1) Links Protein Translation with Oncogenic Metabolism in Leukemia

Nucleophosmin 1 (NPM1) is the most common gene mutated in acute myeloid leukemia (AML). Several putative mechanisms for the role of NPM1 in the generation of AML have been proposed, including acting as a tumor suppressor by promoting the degradation of p53 and sequestration of wild-type NPM1. However, the specificity of the mutation- most commonly a 4 base pair duplication in the C-terminus- suggests that it may be a gain- of- function mutation conferring an unidentified pro-leukemic effect. Using a novel set of leukemia cell lines generated with CRISPR/Cas9 technology in combination with studies in primary hematopoietic cells, we show that mutant NPM1 may promote leukemia by deregulating protein translation with downstream effects on cellular metabolism. Thus, mutant NPM1 may serve as a critical lynchpin to match metabolic precursors and cellular energetics to ribosomal output. The implications of our results suggest that combined metabolic and protein translation inhibition may be a previously unidentified vulnerability in AML with mutated NPM1.CRISPR/Cas9 technology was used to generate a set of isogenic cell lines derived from the heterozygous OCI-AML3 line. Transcriptomic analysis of clones with either parental allele configuration (wild-type and mutant, WTAM) vs. inactivation of the mutant allele (wild-type only, WTO) confirmed that mutant NPM1 regulates HOX genes, as shown in both mouse models and patient samples. Next, the effects of mutant NPM1 on ribosome biogenesis and protein translation were assayed with pulse- chase and static experiments and showed both ribosomal RNA and protein translation were increased in WTAM clones vs. WTO clones. During these investigations, cell size was noted to be increased in WTAM cells. Cell size can be a consequence of the metabolic activity of a cell. Analysis of the rate of glucose consumption and lactate production showed that WTAM cells have increased aerobic glycolysis compared with WTO cells. To test if mutant NPM1 is sufficient to drive both protein translation and aerobic glycolysis in a normal cell, these studies were replicated in normal human hematopoietic stem cells stably expressing either wild-type NPM1 or mutant NPM1.In summary, our results suggest that mutant NPM1 acts through dysregulation of protein translation and participates in metabolic reprogramming required to drive oncogenic transformation. DisclosuresNo relevant conflicts of interest to declare.

Antagonizing Retinoic Acid Receptors Increases Myeloid Cell Production by Cultured Human Hematopoietic Stem Cells

Activities of the retinoic acid receptor (RAR)α and RARγ are important to hematopoiesis. Here, we have investigated the effects of receptor selective agonists and antagonists on the primitive human hematopoietic cell lines KG1 and NB-4 and purified normal human hematopoietic stem cells (HSCs). Agonizing RARα (by AGN195183) was effective in driving neutrophil differentiation of NB-4 cells and this agonist synergized with a low amount (10 nM) of 1α,25-dihydroxyvitamin D3 to drive monocyte differentiation of NB-4 and KG1 cells. Treatment of cultures of human HSCs (supplemented with stem cell factor ± interleukin 3) with an antagonist of all RARs (AGN194310) or of RARα (AGN196996) prolonged the lifespan of cultures, up to 55 days, and increased the production of neutrophils and monocytes. Slowing down of cell differentiation was not observed, and instead, hematopoietic stem and progenitor cells had expanded in number. Antagonism of RARγ (by AGN205728) did not affect cultures of HSCs. Studies of CV-1 and LNCaP cells transfected with RAR expression vectors and a reporter vector revealed that RARγ and RARβ are activated by sub-nM all-trans retinoic acid (EC50–0.3 nM): ~50-fold more is required for activation of RARα (EC50–16 nM). These findings further support the notion that the balance of expression and activity of RARα and RARγ are important to hematopoietic stem and progenitor cell expansion and differentiation.