Faculty Bibliography

Background: The myelodysplastic syndromes (MDS) are disorders of ineffective hematopoiesis that arise in hematopoietic stem cells (HSCs). To better understand the mechanisms underlying MDS pathogenesis and clinical responses to the hypomethylating agent decitabine (DAC), we used single-cell RNA-sequencing to evaluate HSCs from MDS patients treated with DAC as well as from normal young and old controls.
Design(s): HSCs (Lin-CD34+CD38-CD90+CD45RA-) were purified from low-risk MDS patients (n=6) as well as normal controls including cord blood (n=2), young adults (range 21-35; n=2), middle-age adults (range:40-50; n=2) and elderly controls (>65 years old; n=2); paired pre- and post-DAC samples from MDS patients undergoing therapy were evaluated. Using the Fluidigm C1 platform, we captured a total of 1,606 single cells from 26 samples and performed RNA-sequencing (Illumina HiSeq2500,100 bp paired-end reads) Results: Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction revealed that normal and MDS HSCs exhibit unique transactional profiles and confirmed the unique transcriptional features of each of the tested groups (Fig1). Pseudotime ordering showed that MDS HSCs express a gene signature that is independent of age. Intriguingly, mRNAs encoding genes recurrently mutated in myeloid neoplasms and associated with clonal hematopoiesis(e.g. ASXL1, TET2, DNMT3A, PP1MD) were downregulated in the elderly HSCs. HSCs from DAC responders are more similar to normal controls than non-responder HSCs, and are characterized by decreased ribosomal protein expression and increased p53 activation, particularly in non-responders. Comparison of MDS HSCs from responders to non-responder identified 455 DEGs (FDR<0.01) enriched for gene ontology (GO) terms involving "mRNA catabolic process, NMD," "protein localization to the ER," and "translational elongation," with 12 of the top 13 DEGs being ribosomal proteins (RPs). Utilizing the 491 DEGs (FDR< 0.01) in MDS HSCs compared to age-matched controls, we used LASSO regression to identify and validate a 10-gene signature that was able to stratify patients into low, intermediate, and high risk groups and predict overall survival (OS) in a large cohort of MDS patients (GSE19429) (Figure presented)
Conclusion(s): Single-cell RNA sequencing identifies genes that distinguish MDS from normal HSCs. MDS HSCs exhibit transcriptional changes that predict OS as well as clinical responses to DAC therapy. These studies support the importance of HSCs in determining clinical outcomes in MDS

Understanding the mechanisms underlying anti-tumor immunity is pivotal for improving immune-based cancer therapies. Here, we report that growth of BRAF-mutant melanoma cells is inhibited, up to complete rejection, in Siah2^-/- mice. Growth-inhibited tumors exhibit increased numbers of intra-tumoral activated T cells and decreased expression of Ccl17, Ccl22, and Foxp3. Marked reduction in Treg proliferation and tumor infiltration coincide with G1 arrest in tumor infiltrated Siah2^-/- Tregs in vivo or following T cell stimulation in culture, attributed to elevated expression of the cyclin-dependent kinase inhibitor p27, a Siah2 substrate. Growth of anti-PD-1 therapy resistant melanoma is effectively inhibited in Siah2^-/- mice subjected to PD-1 blockade, indicating synergy between PD-1 blockade and Siah2 loss. Low SIAH2 and FOXP3 expression is identified in immune responsive human melanoma tumors. Overall, Siah2 regulation of Treg recruitment and cell cycle progression effectively controls melanoma development and Siah2 loss in the host sensitizes melanoma to anti-PD-1 therapy.

The cell-of-origin of high grade serous ovarian carcinoma (HGSOC) remains controversial, with fallopian tube epithelium (FTE) and ovarian surface epithelium (OSE) both considered candidates. Here, by using genetically engineered mouse models and organoids, we assessed the tumor-forming properties of FTE and OSE harboring the same oncogenic abnormalities. Combined RB family inactivation and Tp53 mutation in Pax8 + FTE caused Serous Tubal Intraepithelial Carcinoma (STIC), which metastasized rapidly to the ovarian surface. These events were recapitulated by orthotopic injection of mutant FTE organoids. Engineering the same genetic lesions into Lgr5 + OSE or OSE-derived organoids also caused metastatic HGSOC, although with longer latency and lower penetrance. FTE- and OSE-derived tumors had distinct transcriptomes, and comparative transcriptomics and genomics suggest that human HGSOC arises from both cell types. Finally, FTE- and OSE-derived organoids exhibited differential chemosensitivity. Our results comport with a dualistic origin for HGSOC and suggest that the cell-of-origin might influence therapeutic response.

Aging hematopoiesis is characterized by increased numbers of immunophenotypic HSCs that exhibit impaired self-renewal and long-term reconstitution potential, both in competitive and noncompetitive settings. We previously demonstrated that normal young mouse HSCs (CD34-CD150+LSK) can be fractionated into subsets based on expression of c-Kit surface expression, with c-KitXX HSCs exhibiting reduced self-renewal and megakaryocytic biased differentiation (Shin et al., 2014). We therefore hypothesized that the expansion of c-KitXX HSCs in old mice could potentially explain the age-related decline in immunophenotypically defined old HSC function. Evaluation of the bone marrow of 24-month-old C57Bl/6 mice revealed that the frequency of c-KitXXHSCs (out of total HSCs) is 1.5-fold higher in old mice than in 3-month old mice (P=0.04), while the frequency of c-KitXX HSCs was 1.5-fold lower in old mice (P=0.007; Fig 1A). This finding is consistent with our previous observation of a megakaryocytic-bias in c-KitXXHSCs, since peripheral blood analysis of old mice revealed a 2.1-fold increase in platelets compared to young mice (p<0.01) (Fig 1B). To test the long-term reconstitution potential of aging HSCs, we competitively transplanted 400 c-KitXX HSCs from 24-month old mice, along with 300,000 competitor bone marrow cells, into lethally irradiated young recipients. Sixteen weeks post-transplantation, mice receiving old c-KitXX HSCs exhibited significantly lower donor peripheral blood chimerism levels compared to old c-KitXX HSC recipients (9.4% vs 57.1%, P=0.02) (Fig 1C). Both old c-KitXXand old c-KitXX HSCs exhibited similar myeloid-reconstituting potential (Fig 1D). Furthermore, mice transplanted with old c-KitXX HSCs exhibited 78% donor HSC chimerism, achieving 6.4-fold higher chimerism levels than mice transplanted with old c-KitXX HSCs, this was comparable to the differences observed with young c-KitXX transplanted HSCs (Fig 1E). To quantify the self-renewal capacity of old HSCs, we calculated the "self-renewal quotient" (Challen et al., 2010). This analysis showed that the self-renewal potential in old c-KitXX HSCs were 0.8 and 7.8 respectively, indicating higher self-renewal potential in c-KitXX HSCs (Fig 1F). Collectively, these data suggest that myeloid-biased differentiation is an age-associated change in hematopoiesis that may not be associated with decreased self-renewal in all HSCs. To gain mechanistic insights underlying these qualitative differences, we interrogated transcriptional profiles of microarray data from c-KitXX HSCs, to identify potential pathways critical for HSC maintenance. Gene Ontology and pathway analyses showed several differentially expressed pathways between c-KitXXHSCs, of which genes related to protein translation and mitochondrial activity was significantly enriched in c-KitXX HSCs (Fig 1G). Given the underrepresentation of translation-related genes in c-KitXX HSCs, we tested whether they exhibit reduced global translation using OP-Puro incorporation assays. These studies confirmed that old c-KitXX HSCs show lower global translation levels than c-KitXXHSCs (Fig 1H). Overall, our studies demonstrate functional heterogeneity among old HSCs and identify a novel strategy to identify old HSCs with preserved self-renewal and long-term reconstitution capacity. The ability to identify and prospectively fractionate old HSCs offers a novel approach to investigate the molecular mechanisms underlying HSC aging. Figure legend. (A) Frequency of c-KitXXor c-KitXX HSCs was assessed by flow cytometry. (B) Circulating platelet numbers were assessed using a Hemavet counter. Competitive transplants of old c-KitXX and c-KitXX HSCs into lethally irradiated recipients (C-F). Donor chimerism (C) and lineage potential (D) was evaluated in the peripheral blood of primary recipients. Bone marrow was analyzed at 16 weeks, for donor-derived HSC chimerism (E) and self-renewal quotient (F). (G) Enrichment plots comparing microarray data generated from c-KitXX HSCs, using pathways translation-related gene sets. (H) OP-Puro incorporation assays in 24-month old mice. Results are representative of three independent experiments, and shown as mean +/- SEM. n = 4-5 mice. *, P < 0.05; **, P < 0.01. [Formula presented] Disclosures: No relevant conflicts of interest to declare.XXCopyright

Multifocal breast cancer (MFBC), ductal type, has been hypothesized to arise by one of two mechanisms: either through intramammary/intralymphatic spread from a single index tumor (MBC-1), or as multiple independent tumors with each focus carrying its corresponding ductal carcinoma in-situ (MBC-2). In order to improve our understanding of MFBC pathogenesis, we employed laser capture microdissection coupled with whole-exome sequencing to study clonal origin in MFBC. We selected three cases of MBC-1 (C1 to C3) and MBC-2 (C4 to C6) and analyzed three foci from each case. MBC-1 cases were histologically similar and showed a strong predilection for satellite foci, vascular invasion and nodal metastasis when compared to MBC-2. Our bioinformatics approach provided strong evidence for clonal relationships in MBC-1, as demonstrated by distinct clusters of genes conserved across all tumor foci. Conversely, no gene clusters were shared across all the foci in MBC-2, suggesting multiple independent tumors. These findings provide further support for the two distinct pathogenetic mechanisms in MFBC.

Recurrent somatic missense mutations in histone H3 genes have been identified in subsets of pediatric cancers. H3K36 histone mutations have recently been recognized as oncogenic drivers in rare subsets of malignant soft tissue sarcomas but have not been reported in histiocytic neoplasms. Currently, the histological and molecular spectrum, as well as the clinical behavior of H3K36-mutant soft tissue malignancies, is largely unknown. We describe a pediatric patient with a HIST1H3B K36I-mutant histiocytic tumor arising in the skull. After the failure of upfront therapy for histiocytosis and development of widely disseminated metastatic disease, the patient had an exceptional response to empiric chemotherapy and remains in complete disease remission for more than 5 years. Our report expands the histological spectrum of H3K36M/I-mutant soft tissue malignancies to histiocytic neoplasms and indicates that multiagent sarcoma-like chemotherapy can be highly effective even in the setting of widely disseminated metastatic disease.

The response to systemic infection and injury requires the rapid adaptation of hematopoietic stem cells (HSCs), which proliferate and divert their differentiation toward the myeloid lineage. Significant interest has emerged in understanding the signals that trigger the emergency hematopoietic program. However, the mechanisms that halt this response of HSCs, which is critical to restore homeostasis, remain unknown. Here we reveal that the E3 ubiquitin ligase Speckle-type BTB-POZ protein (SPOP) restrains the inflammatory activation of HSCs. In the absence of Spop, systemic inflammation proceeded in an unresolved manner, and the sustained response in the HSCs resulted in a lethal phenotype reminiscent of hyper-inflammatory syndrome or sepsis. Our proteomic studies decipher that SPOP restricted inflammation by ubiquitinating the innate signal transducer myeloid differentiation primary response protein 88 (MYD88). These findings unearth an HSC-intrinsic post-translational mechanism that is essential for reestablishing homeostasis after emergency hematopoiesis.

Unconventional T lymphocyte populations are emerging as important regulators of tumor immunity. Despite this, the role of TCRαβ+CD4-CD8-NK1.1- innate αβ T-cells (iαβTs) in pancreatic ductal adenocarcinoma (PDA) has not been explored. We found that iαβTs represent ~10% of T-lymphocytes infiltrating PDA in mice and humans. Intra-tumoral iαβTs express a distinct TCR-repertoire and profoundly immunogenic phenotype compared to their peripheral counterparts and conventional lymphocytes. iαβTs comprised ~75% of the total intra-tumoral IL-17+ cells. Moreover, iαβT cell adoptive transfer is protective in both murine models of PDA and human organotypic systems. We show iαβT cells induce a CCR5-dependent immunogenic macrophage reprogramming, thereby enabling marked CD4+ and CD8+ T cell expansion/activation and tumor protection. Collectively, iαβTs govern fundamental intra-tumoral crosstalk between innate and adaptive immune populations and are attractive therapeutic targets.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.