Faculty Bibliography

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.

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.

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.

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As with most cancer types, there remains a subset of B-cell acute lymphoblastic leukaemia (B-ALL) patients who will relapse and succumb to therapy-resistant disease. It is believed that tumour heterogeneity underpins therapy failure leading to a Darwinian model of clonal evolution, however, such studies do not account for the role of the bone marrow microenvironment in supporting leukaemia survival, progression and escape from treatment. Here, we perform single-cell RNA-Sequencing (scRNA-Seq) to generate a comprehensive map of the primary human B-ALL bone marrow immune microenvironment throughout three distinct stages of the human leukemic disease process: diagnosis, remission and relapse. These studies show extensive re-modelling of the immune microenvironment composition and cell-to-cell interactions throughout the course conventional chemotherapy, and uncover a role for inflammatory leukaemia-associated monocytes in promoting B-ALL pathogenesis in vivo. These monocytic subsets are predictive of Ph+ B-ALL patient event-free survival and when targeted in B-ALL animal models, lead to prolonged disease remission. Our profiling of the human B-ALL bone marrow immune microenvironment provides a greater understanding of the potential extrinsic regulators of B-ALL survival and may highlight previously unknown environmental factors influencing immune-based treatment approaches to high-risk B-ALL.

Pancreatic adenocarcinoma (PDA) is an aggressive disease driven by oncogenic KRAS and characterized by late diagnosis and therapeutic resistance. Here we show that deletion of the ataxia-telangiectasia group D-complementing (Atdc) gene, whose human homolog is up-regulated in the majority of pancreatic adenocarcinoma, completely prevents PDA development in the context of oncogenic KRAS. ATDC is required for KRAS-driven acinar-ductal metaplasia (ADM) and its progression to pancreatic intraepithelial neoplasia (PanIN). As a result, mice lacking ATDC are protected from developing PDA. Mechanistically, we show ATDC promotes ADM progression to PanIN through activation of β-catenin signaling and subsequent SOX9 up-regulation. These results provide new insight into PDA initiation and reveal ATDC as a potential target for preventing early tumor-initiating events.

The bone marrow microenvironment has a key role in regulating haematopoiesis, but its molecular complexity and response to stress are incompletely understood. Here we map the transcriptional landscape of mouse bone marrow vascular, perivascular and osteoblast cell populations at single-cell resolution, both at homeostasis and under conditions of stress-induced haematopoiesis. This analysis revealed previously unappreciated levels of cellular heterogeneity within the bone marrow niche and resolved cellular sources of pro-haematopoietic growth factors, chemokines and membrane-bound ligands. Our studies demonstrate a considerable transcriptional remodelling of niche elements under stress conditions, including an adipocytic skewing of perivascular cells. Among the stress-induced changes, we observed that vascular Notch delta-like ligands (encoded by Dll1 and Dll4) were downregulated. In the absence of vascular Dll4, haematopoietic stem cells prematurely induced a myeloid transcriptional program. These findings refine our understanding of the cellular architecture of the bone marrow niche, reveal a dynamic and heterogeneous molecular landscape that is highly sensitive to stress and illustrate the utility of single-cell transcriptomic data in evaluating the regulation of haematopoiesis by discrete niche populations.

Microglandular adenosis (MGA) is a rare breast lesion reported to be associated with invasive carcinoma in up to 20-30% of cases, and has been proposed as a non-obligate precursor to basal-like breast cancers. We identified a case of matrix-producing metaplastic carcinoma with morphologic and immunohistochemical evidence of progression from MGA to atypical MGA (AMGA), carcinoma in situ (CIS) and invasive carcinoma. We performed whole exome sequencing of each component (MGA, AMGA, CIS and cancer) to characterize the mutational landscape of these foci. There was significant copy number overlap between all foci, including a segmental amplification of the CCND1 locus (partial chromosome 11 trisomy) and MYC (8q24.12-13). Using a bioinformatics approach, we were able to identify three putative mutational clusters and recurrent, stop-gain non-synonymous mutations in both ZNF862 and TP53 that were shared across all foci. Finally, we identified a novel deleterious splice-acceptor site mutation of chr5:5186164G>T (chromosome 5p15) encoding the gene, ADAMTS16, in the invasive component.

BACKGROUND:Therapies targeting anti-tumor T-cell responses have proven successful in the treatment of a variety of malignancies. However, as most patients still fail to respond, approaches to augment immunotherapeutic efficacy are needed. Here, we investigated the ability of histone deacetylase 6 (HDAC6)-selective inhibitors to decrease immunosuppression and enhance immune function of melanoma patient T-cells in ex vivo cultures. METHODS:T-cells were harvested from peripheral blood or tumor biopsies of metastatic melanoma patients and cultured in the presence of pan-, class-specific or class-selective histone deacetylase (HDAC) inhibitors. Changes in cytokine production were evaluated by Luminex and intracellular flow cytometry staining. Expression of surface markers, transcription factors, protein phosphorylation, and cell viability were assessed by flow cytometry. Changes in chromatin structure were determined by ATAC-seq. RESULTS:T-cell viability was impaired with low doses of pan-HDAC inhibitors but not with specific or selective HDAC inhibitors. The HDAC6-selective inhibitors ACY-1215 (ricolinostat) and ACY-241 (citarinostat) decreased Th2 cytokine production (i.e. IL-4, IL-5, IL-6, IL-10 and IL-13). Expansion of peripheral blood T-cells from melanoma patients in the presence of these inhibitors resulted in downregulation of the Th2 transcription factor GATA3, upregulation of the Th1 transcription factor T-BET, accumulation of central memory phenotype T-cells (CD45RA-CD45RO + CD62L + CCR7+), reduced exhaustion-associated phenotypes (i.e. TIM3 + LAG3 + PD1+ and EOMES+PD1+), and enhanced killing in mixed lymphocyte reactions. The frequency, FOXP3 expression, and suppressive function of T regulatory cells (Tregs) were decreased after exposure to ACY-1215 or ACY-241. Higher frequencies of T-cells expressing CD107a + IFNγ+ and central memory markers were observed in melanoma tumor-infiltrating lymphocytes (TIL), which persisted after drug removal and further expansion. After ACY-1215 treatment, increased chromatin accessibility was observed in regions associated with T-cell effector function and memory phenotypes, while condensed chromatin was found in regions encoding the mTOR downstream molecules AKT, SGK1 and S6K. Decreased phosphorylation of these proteins was observed in ACY-1215 and ACY-241-treated T-cells. AKT- and SGK1-specific inhibition recapitulated the increase in central memory frequency and decrease in IL-4 production, respectively, similar to the observed effects of HDAC6-selective inhibition. CONCLUSIONS:HDAC6-selective inhibitors augmented melanoma patient T-cell immune properties, providing a rationale for translational investigation assessing their potential clinical efficacy.