Faculty Bibliography

B cell acute lymphoblastic leukemia (B-ALL) blasts hijack the bone marrow (BM) microenvironment to form chemoprotective leukemic BM "niches," facilitating chemoresistance and, ultimately, disease relapse. However, the ability to dissect these evolving, heterogeneous interactions among distinct B-ALL subtypes and their varying BM niches is limited with current in vivo methods. Here, we demonstrated an in vitro organotypic "leukemia-on-a-chip" model to emulate the in vivo B-ALL BM pathology and comparatively studied the spatial and genetic heterogeneity of the BM niche in regulating B-ALL chemotherapy resistance. We revealed the heterogeneous chemoresistance mechanisms across various B-ALL cell lines and patient-derived samples. We showed that the leukemic perivascular, endosteal, and hematopoietic niche-derived factors maintain B-ALL survival and quiescence (e.g., CXCL12 cytokine signal, VCAM-1/OPN adhesive signals, and enhanced downstream leukemia-intrinsic NF-κB pathway). Furthermore, we demonstrated the preclinical use of our model to test niche-cotargeting regimens, which may translate to patient-specific therapy screening and response prediction.

Oral cancer patients experience pain at the site of the primary cancer. Patients with metastatic oral cancers report greater pain. Lack of pain identifies patients at low risk of metastasis with sensitivity = 0.94 and negative predictive value = 0.89. In the same cohort, sensitivity and negative predictive value of depth of invasion, currently the best predictor, were 0.95 and 0.92, respectively. Cancer pain is attributed to cancer-derived mediators that sensitize neurons and is associated with increased neuronal density. We hypothesized that pain mediators would be overexpressed in metastatic cancers from patients reporting high pain. We identified 40 genes overexpressed in metastatic cancers from patients reporting high pain (n=5) compared to N0 cancers (n=10) and normal tissue (n=5). The genes are enriched for functions in extracellular matrix organization and angiogenesis. They have oncogenic and neuronal functions and are reported in exosomes. Hierarchical clustering according to expression of neurotrophic and axon guidance genes also separated cancers according to pain and nodal status. Depletion of exosomes from cancer cell line supernatant reduced nociceptive behavior in a paw withdrawal assay, supporting a role for exosomes in cancer pain. The identified genes and exosomes are potential therapeutic targets for stopping cancer and attenuating pain.

A subset of B cell acute lymphoblastic leukemia (B-ALL) patients will relapse and succumb to therapy-resistant disease. The bone marrow microenvironment may support B-ALL progression and treatment evasion. Utilizing single-cell approaches, we demonstrate B-ALL bone marrow immune microenvironment remodeling upon disease initiation and subsequent re-emergence during conventional chemotherapy. We uncover a role for non-classical monocytes in B-ALL survival, and demonstrate monocyte abundance at B-ALL diagnosis is predictive of pediatric and adult B-ALL patient survival. We show that human B-ALL blasts alter a vascularized microenvironment promoting monocytic differentiation, while depleting leukemia-associated monocytes in B-ALL animal models prolongs disease remission in vivo. Our profiling of the B-ALL immune microenvironment identifies extrinsic regulators of B-ALL survival supporting new immune-based therapeutic approaches for high-risk B-ALL treatment.

Next-generation sequencing has enabled genetic and genomic characterization of melanoma to an unprecedent depth. However, the high mutational background plus the limited deep-coverage whole-genome sequencing performed on cutaneous melanoma samples, make difficult the identification of novel driver mutations. We sought to explore the somatic mutation portfolio in exonic and gene regulatory regions in human melanoma samples, for which we performed targeted sequencing of tumors and matched germline DNA samples from 89 melanoma patients, identifying known and novel recurrent mutations. Two recurrent mutations found in the RPS27 promoter associated with decreased RPS27 mRNA levels in vitro. Data mining and IHC analyses revealed a bimodal pattern of RPS27 expression in melanoma, with RPS27-low patients displaying worse prognosis. In vitro characterization of RPS27-high and -low melanoma cell lines, as well as loss-of-function experiments, demonstrated that high RPS27 status provides increased proliferative and invasive capacities, while low RPS27 confers survival advantage in low-attachment and resistance to therapy. Additionally, we demonstrate that 10 other cancer types harbor bimodal RPS27 expression and in those, similarly to melanoma, RPS27-low expression associates with worse clinical outcomes. RPS27 promoter mutation could thus represent a mechanism of gene expression modulation in melanoma patients, which may have prognostic and predictive implications.

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