Adipocyte depots throughout the body are physiologically and molecularly distinct. With age, adipocytes increase in and around aged thymi. Yet thymic adipocytes lack molecular characterization. We developed methods to isolate adipocyte nuclei from mouse thymi. Single-nucleus multi-omic analysis of male and female mice aged 4-9 months reveals that thymic adipocytes are heterogeneous, with two distinct populations. One subpopulation harbors a transcription and chromatin signature consistent with beige fat. Another subpopulation resembles classic white adipose tissue and expresses genes associated with epithelial-to-mesenchymal transition (EMT). Analysis of differentially open chromatin identifies binding sites for Foxn1 and HIF-1α/Arnt in the white adipose population, consistent with a thymic epithelial and/or hypoxic origin for these cells. Immunofluorescence confirmed the expression of UCP1 protein in cells in subcapsular cortical regions of the thymic parenchyma. This resource reveals a complex milieu of thymic adipocytes and identifies multiple avenues for probing their ontogeny, dynamics, and functional significance.

The 1,287 genes targeted by the shRNA of the leukemia library.

Next generation sequencing for 52 myeloid malignancies-related genes.

Patient sample information.

Ranked list of 1,287 genes from shRNA library screen in primary AML cells. The 1,287 genes assessed with an shRNA library screen were sorted by the second highest percentile fold change present in 2 shRNA and across 2 samples, with the 34 genes showing a fold change in the top 2 percent in more than 2 samples listed first.

Standard hematologic parameters in SIRT5-/- mice and SIRT5+/+ littermates.

All supplemental figures and legends.

Abstract We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML. Significance: Reducing SIRT5 activity is detrimental to the survival of AML cells regardless of genotype, yet well tolerated by healthy hematopoietic cells. In mouse models, disrupting SIRT5 inhibits AML progression. SIRT5 controls several metabolic pathways that are required for leukemia cell survival. These results identify SIRT5 as a therapeutic target in AML. See related commentary by Li and Melnick, p. 198.

The chronic phase of chronic myeloid leukemia (CP-CML) is characterized by excessive production of maturating myeloid cells. As CML stem/progenitor cells (LSPCs) are poised to cycle and differentiate, LSPCs must balance conservation and differentiation to avoid exhaustion, similar to normal hematopoiesis under stress. Since BCR-ABL1 tyrosine kinase inhibitors (TKIs) eliminate differentiating cells, but spare BCR-ABL1-independent LSPCs, understanding the mechanisms that regulate LSPC differentiation may inform strategies to eliminate LSPCs. Upon performing a meta-analysis of published CML transcriptomes, we discovered that low expression of the MS4A3 transmembrane protein is a universal characteristic of LSPC quiescence, BCR-ABL1 independence, and transformation to blast phase. Several mechanisms are involved in suppressing MS4A3, including aberrant methylation and a MECOM-C/EBPε axis. Contrary to previous reports, we find that MS4A3 does not function as a G1/S phase inhibitor, but promotes endocytosis of common β chain (βc) cytokine receptors upon GM-CSF/IL-3 stimulation, enhancing downstream signaling and cellular differentiation. This suggests that LSPCs downregulate MS4A3 to evade βc cytokine-induced differentiation and maintain a more primitive, TKI-insensitive state. Accordingly, knockdown or deletion of MS4A3/Ms4a3 promotes TKI resistance and survival of CML cells ex vivo and enhance leukemogenesis in vivo, while targeted delivery of exogenous MS4A3 protein promotes differentiation. These data support a model in which MS4A3 governs response to differentiating myeloid cytokines, providing a unifying mechanism for the differentiation block characteristic of CML quiescence and blast phase CML. Promoting MS4A3 re-expression or delivery of ectopic MS4A3 may help eliminating LSPCs in vivo.

Abstract Standard of care for AML includes chemotherapy and stem cell transplant, with 5-year survival rates <30%. We sought to identify genes critical to AML cells, irrespective of mutational status, and performed an shRNA screen targeting 1,287 genes on 12 AML patient samples. This screen identified Sirtuin 5 (SIRT5) as a top candidate. SIRT5 is the only known enzyme with desuccinylase, demalonylase, and/or deglutarylase activity and we are the first to report the dependence of AML cells on SIRT5. Next, we stably transduced a panel of AML cell lines with doxycycline (dox)-inducible shSIRT5 (dox-shSIRT5). SIRT5 knockdown (KD) strongly inhibited cell growth, colony formation and increased apoptosis in 15/22 lines (SIRT5-dependent), while 7/22 lines were SIRT5-independent. SIRT5 dependence did not correlate with AML-related mutations nor basal SIRT5 expression. SIRT5 KD in primary AML samples (N=25) revealed a therapeutic window (~50% reduction), with no effect in CB samples (N=5). We examined the requirement of SIRT5 in vivo using three mouse models of leukemia. In a xenograft model with AML cell lines, SIRT5 KD indefinitely prolonged survival of mice injected with SIRT5-dependent cells with no sign of leukemia. Bone marrow transplant with transduced (MLL-AF9 or BCR-ABL1) SIRT5 null cells showed reduced leukemia cell burden and splenomegaly, and significantly prolonged survival. FLT3-ITD-driven disease was also blunted by the absence of SIRT5 in a genetic knockout mouse model. Mechanically, SIRT5 KD profoundly reduced oxidative phosphorylation (OXPHOS) and glycolysis. Additionally, SIRT5 KD increased mitochondrial superoxide selectively in annexin V-negative, SIRT5-dependent cells. Concomitant, ectopic expression of SOD2 abrogated the increase in superoxide, rescued cells from apoptosis, and rescued the colony formation deficit. Untargeted metabolomics revealed RNA charging and alanine and serine metabolism as top metabolic pathways regulated by SIRT5, with glutaminase (GLS) and α-ketoglutarate identified as potential upstream regulators. Metabolic tracing experiments with [13C5,15N2]-glutamine confirmed disrupted glutamine metabolism in SIRT5-dependent cells. Together, these results indicate that SIRT5 is required to regulate glutamine flux to sustain redox homeostasis and/or anabolism. NRD167, a novel SIRT5 inhibitor, was used to target SIRT5 in AML. NRD167 reduced cell proliferation, induced apoptosis, and reduced OXPHOS in SIRT5-dependent but not SIRT5-independent cells. NRD167 inhibited colony formation from AML patient samples, but not in CB samples. An AML patient-derived xenograft model trended toward prolonged survival following ex vivo treatment with NRD167. Our data suggest that the majority of AML samples are dependent on SIRT5 and that inhibition preferentially targets AML cells, implicating SIRT5 as a therapy target in AML. Citation Format: Dongqing Yan, Anca Franzini, Anthony D. Pomicter, Brayden J. Halverson, Orlando Antelope, Clinton C. Mason, Jonathan M. Ahmann, Anna V. Senina, Courtney L. L. Jones, Matthew S. Zabriskie, Hein Than, Michael J. Xiao, Alexandria van Scoyk, Ami B. Patel, William L. L. Heaton, Shawn C. Owen, Joshua L. Andersen, Christina M. Egbert, Julie A. Reisz, Angelo D'Alessandro, James E. Cox, Kevin C. Gantz, Hannah M. Redwine, Siddharth M. Iyer, Jamshid S. Khorashad, Nima Rajabi, Christian A. Olsen, Thomas O'Hare, Michael W. Deininger. A critical role for SIRT5 in acute myeloid leukemia metabolism [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 LB109.

The authors describe recurrent novel insertion/deletion mutations in the JH2 domain of JAK2 occurring in patients with eosinophilia as a prominent feature of their myeloproliferative neoplasms. Remarkably, 2 of the patients with a specific mutation (Leu583-Ala586DelInsSer) meet the criteria for both chronic eosinophilic leukemia and polycythemia vera, suggesting that this may be a distinct overlap syndrome.

Chronic myelomonocytic leukemia (CMML) is an aggressive myeloid neoplasm of older individuals characterized by persistent monocytosis. Somatic mutations in CMML are heterogeneous and only partially explain the variability in clinical outcomes. Recent data suggest that cardiovascular morbidity is increased in CMML and contributes to reduced survival. Clonal hematopoiesis of indeterminate potential (CHIP), the presence of mutated blood cells in hematologically normal individuals, is a precursor of age-related myeloid neoplasms and associated with increased cardiovascular risk. To isolate CMML-specific alterations from those related to aging, we performed RNA sequencing and DNA methylation profiling on purified monocytes from CMML patients and from age-matched (old) and young healthy controls. We found that the transcriptional signature of CMML monocytes is highly proinflammatory, with upregulation of multiple inflammatory pathways, including tumor necrosis factor and interleukin (IL)-6 and -17 signaling, whereas age per se does not significantly contribute to this pattern. We observed no consistent correlations between aberrant gene expression and CpG island methylation, suggesting that proinflammatory signaling in CMML monocytes is governed by multiple and complex regulatory mechanisms. We propose that proinflammatory monocytes contribute to cardiovascular morbidity in CMML patients and promote progression by selection of mutated cell clones. Our data raise questions of whether asymptomatic patients with CMML benefit from monocyte-depleting or anti-inflammatory therapies.

Acute myeloid leukemia (AML) is an aggressive hematopoietic neoplasm with five-year overall survival rates <30% on standard of care therapy. Relapse is common and thought to originate from AML stem cells that survive in the protective bone marrow (BM) microenvironment. Next generation sequencing (NGS) has revealed the somatic mutation spectrum of AML and the approval of midostaurin for FLT3 ITD- and enasidenib for IDH2 -mutated AML is evidence that the molecular knowledge is being translated clinically.

Chronic myelomonocytic leukemia (CMML) is a genetically heterogeneous hematopoietic stem cell disorder that combines features of a myelodysplastic syndrome and a myeloproliferative neoplasm and exhibits a strong bias towards older age. The prognosis of CMML is poor, with overall survival of less than 3 years in most studies, however recurrent somatic mutations explain only 15-24% of the clinical heterogeneity of CMML (Elena C. et al. Blood 128:1408-17, 2016). The extreme skewing of the CMML age distribution suggests that CMML reflects the malignant conversion of the myelomonocytic-biased differentiation characteristic of an aged hematopoietic system. We hypothesized that separating the contribution of the normal aging process from bona fide CMML-specific alterations will improve the molecular characterization and biological understanding of CMML. We decided to focus on monocytes as the phenotypic minimal common denominator of genetically heterogeneous diseases. CD14+ monocytes were sorted from the blood of untreated CMML patients (N=12, median age 77 years, range 61-90), age-matched healthy controls (old controls: N=12, median age 68 years, range 62-74) and young healthy controls (young controls: N=16, median age 29 years, range 24-44) and subjected to RNA sequencing and DNA methylation profiling. Differentially expressed genes in CMML monocytes compared to healthy controls were identified with DESeq2 using a 1% false discovery rate (FDR) and a fold-change cutoff set at >│2│ (Figure 1A). We identified the 2480 CMML-specific genes by subtracting all genes with significant differences in the young controls vs. old controls comparison from the CMML vs. old controls comparison. The top-25 most significantly upregulated genes (Figure 1B) included transcription factors, TNFα signaling genes, genes that regulate genomic stability, and genes involved in apoptosis. The most significantly downregulated transcripts were genes involved in response to DNA damage, RNA binding, monocyte differentiation and mediators of inflammatory process.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a poor prognosis. FLT3 internal tandem duplications (ITDs) are found in ~30% of AML patients and are associated with inferior survival. Addition of FLT3 tyrosine kinase inhibitors (TKIs) such as midostaurin improve survival of AML treated with cytotoxic chemotherapy. However, relapse rates remain unacceptably high. Protection by stroma-derived survival signals is thought to enable survival of leukemia initiating cells (LICs) in the presence of FLT3 TKIs, providing a reservoir for subsequent overt relapse. We previously reported that HS-5 conditioned medium (CM) rescued FLT3-ITD positive AML cell lines and primary cells from the effects of the 2nd generation FLT3 TKI AC220 (quizartinib) as compared to cells cultured in regular medium (RM). Earlier work from our lab and others has demonstrated that HS-5 CM increases pSTAT3Y705 in chronic myeloid leukemia cells treated with BCR-ABL1 TKIs, leading to TKI resistance, while pSTAT5Y694 levels remain under the control of the BCR-ABL1 kinase (Eiring et al, Leukemia, 2015). Similar to BCR-ABL1, FLT3 signaling results in potent pSTAT5Y694 activation. We hypothesized that STAT3 and/or STAT5 activation contributes to stroma-mediated protection of FLT3-ITD+ AML cells upon FLT3 inhibition with AC220.

Chronic myelomonocytic leukemia (CMML) is a clonal hematologic neoplasm that combines features of myelodysplasia and myeloproliferative disorder, and carries a poor prognosis due to progression to acute myeloid leukemia or complications of cytopenias. Hematopoietic stem cell transplantation is the only curative option, but many patients are ineligible due to comorbidities and old age. Previous genomic analyses reveal recurrent somatic driver mutations in a variety of genes, including many epigenetic regulators. Hypomethylating agents (HMAs) such as 5-azacitidine induce hematologic responses in approximately 40% of CMML patients, but most patients ultimately experience disease progression.

JAK2 fusions reported in hematologic malignancies are often associated with eosinophilia. In addition to JAK2 fusions such as PCM1-JAK2, BCR-JAK2 and ETV6-JAK2, the JAK2V617Fpoint mutant has been recurrently observed in patients with hypereosinophilic syndrome (HES), occurring in ~4% of patients with hypereosinophilia of unknown significance (Schwaab, Am J Hematol, 2015).