Faculty Bibliography

PURPOSE/OBJECTIVE:Increasing tumor-infiltrating lymphocytes (TILs) is associated with higher rates of pathologic complete response (pCR) to neoadjuvant therapy (NAT) in patients with triple-negative breast cancer (TNBC). However, the presence of TILs does not consistently predict pCR, therefore, the current study was undertaken to more fully characterize the immune cell response and its association with pCR. EXPERIMENTAL DESIGN/METHODS:We obtained pre-treatment core needle biopsies from 105 patients with stage I-III TNBC enrolled in ARTEMIS (NCT02276443) who received NAT from 10/22/2015 through 7/24/2018. The tumor-immune microenvironment was comprehensively profiled by performing T-cell receptor (TCR) sequencing, PD-L1 immunohistochemistry, multiplex immunofluorescence and RNA sequencing on pretreatment tumor samples. The primary endpoint was pathological response to NAT. RESULTS:ratio (median=14.70 vs 8.20, p=0.0128), and closer spatial proximity of T-cells to tumor cells (median=19.26µm vs 21.94µm, p=0.0169) were associated with pCR. In a multivariable model, the closer spatial proximity of T-cells to tumor cells and PD-L1 expression enhanced prediction of pCR when considered in conjunction with clinical stage. CONCLUSIONS:In patients receiving NAT for TNBC, deep immune profiling through detailed phenotypic characterization and spatial analysis can improve prediction of pCR in patients receiving NAT for TNBC when considered with traditional clinical parameters.

Despite remarkable responses to cancer immunotherapy in a subset of patients, many patients remain resistant to therapies. It is now clear that elevated levels of tumor-infiltrating T cells as well as a systemic anti-tumor immune response are requirements for successful immunotherapies. However, the tumor microenvironment imposes an additional resistance mechanism to immunotherapy. We have developed a practical and improved strategy for cancer immunotherapy using an oncolytic virus and anti-OX40. This strategy takes advantage of a preexisting T cell immune repertoire in vivo, removing the need to know about present tumor antigens. We have shown in this study that the replication-deficient oncolytic Sindbis virus vector expressing interleukin-12 (IL-12) (SV.IL12) activates immune-mediated tumor killing by inducing OX40 expression on CD4 T cells, allowing the full potential of the agonistic anti-OX40 antibody. The combination of SV.IL12 with anti-OX40 markedly changes the transcriptome signature and metabolic program of T cells, driving the development of highly activated terminally differentiated effector T cells. These metabolically reprogrammed T cells demonstrate enhanced tumor infiltration capacity as well as anti-tumor activity capable of overcoming the repressive tumor microenvironment. Our findings identify SV.IL12 in combination with anti-OX40 to be a novel and potent therapeutic strategy that can cure multiple types of low-immunogenic solid tumors.

Metastasis is a major cause of cancer mortality. We generated an autochthonous transgenic mouse model whereby conditional expression of MYC and Twist1 enables hepatocellular carcinoma (HCC) to metastasize in >90% of mice. MYC and Twist1 cooperate and their sustained expression is required to elicit a transcriptional program associated with the activation of innate immunity, through secretion of a cytokinome that elicits recruitment and polarization of tumor associated macrophages (TAMs). Systemic treatment with Ccl2 and Il13 induced MYC-HCCs to metastasize; whereas, blockade of Ccl2 and Il13 abrogated MYC/Twist1-HCC metastasis. Further, in 33 human cancers (n = 9502) MYC and TWIST1 predict poor survival (p=4.3×10^-10), CCL2/IL13 expression (p<10^-109) and TAM infiltration (p<10^-96). Finally, in the plasma of patients with HCC (n = 25) but not cirrhosis (n = 10), CCL2 and IL13 were increased and IL13 predicted invasive tumors. Therefore, MYC and TWIST1 generally appear to cooperate in human cancer to elicit a cytokinome that enables metastasis through crosstalk between cancer and immune microenvironment.

Recurrent Respiratory Papillomatosis (RRP) is a rare disease of the aerodigestive tract caused by the Human Papilloma Virus (HPV) that manifests as profoundly altered phonatory and upper respiratory anatomy. Current therapies are primarily symptomatic; enhanced insight regarding disease-specific biology of RRP is critical to improved therapeutics for this challenging population. Multiplex PCR was performed on oral rinses collected from twenty-three patients with adult-onset RRP every three months for one year. Twenty-two (95.6%) subjects had an initial HPV positive oral rinse. Of those subjects, 77.2% had an additional positive oral rinse over 12 months. A subset of rinses were then compared to tissue samples in the same patient employing HPViewer to determine HPV subtype concordance. Multiple HPV copies (60-787 per human cell) were detected in RRP tissue in each patient, but a single dominant HPV was found in individual samples. These data confirm persistent oral HPV infection in the majority of patients with RRP. In addition, three novel HPV6 isolates were found and identical HPV strains, at very low levels, were identified in oral rinses in two patients suggesting potential HPV subtype concordance. Finally, somatic heteroplasmic mtDNA mutations were observed in RRP tissue with 1.8 mutations per sample and two nonsynonymous variants. These data provide foundational insight into both the underlying pathophysiology of RRP, but also potential targets for intervention in this challenging patient cohort.

T regulatory (Treg) cells maintain immunological tolerance and organ homeostasis. Activated Treg cells differentiate into effector Treg subsets that acquire tissue-specific functions. Ca²⁺ influx via Ca²⁺ release-activated Ca²⁺ (CRAC) channels formed by STIM and ORAI proteins is required for the thymic development of Treg cells, but its function in mature Treg cells remains unclear. Here we show that deletion of Stim1 and Stim2 genes in mature Treg cells abolishes Ca²⁺ signaling and prevents their differentiation into follicular Treg and tissue-resident Treg cells. Transcriptional profiling of STIM1/STIM2-deficient Treg cells reveals that Ca²⁺ signaling regulates transcription factors and signaling pathways that control the identity and effector differentiation of Treg cells. In the absence of STIM1/STIM2 in Treg cells, mice develop a broad spectrum of autoantibodies and fatal multiorgan inflammation. Our findings establish a critical role of CRAC channels in controlling lineage identity and effector functions of Treg cells.

Mutational inactivation of ATRX (α-thalassemia mental retardation X-linked) represents a defining molecular alteration in large subsets of malignant glioma. Yet the pathogenic consequences of ATRX deficiency remain unclear, as do tractable mechanisms for its therapeutic targeting. Here we report that ATRX loss in isogenic glioma model systems induces replication stress and DNA damage by way of G-quadruplex (G4) DNA secondary structure. Moreover, these effects are associated with the acquisition of disease-relevant copy number alterations over time. We then demonstrate, both in vitro and in vivo, that ATRX deficiency selectively enhances DNA damage and cell death following chemical G4 stabilization. Finally, we show that G4 stabilization synergizes with other DNA-damaging therapies, including ionizing radiation, in the ATRX-deficient context. Our findings reveal novel pathogenic mechanisms driven by ATRX deficiency in glioma, while also pointing to tangible strategies for drug development.

Mutational inactivation of ATRX (a-thalassemia mental retardation X-linked) represents a defining molecular alteration in large subsets of malignant glioma. ATRX encodes a chromatin binding protein widely implicated in epigenetic regulation and remodeling. However, multiple studies have also associated its loss in cancer with replication stress, DNA damage, and copy number alterations (CNAs). The mechanisms by which ATRX deficiency drives this global genomic instability remain unclear. Here we report that ATRX inactivation in isogenic glioma model systems induces replication stress and DNA damage by promoting the formation of deleterious G-quadruplex (G4) secondary structure in DNA. Moreover, these effects are associated with the acquisition of disease-relevant CNAs over time. Prior work has linked G4s with genomic instability as well as CNAs in cancer. We then demonstrate, both in vitro and in vivo, that chemical G4 stabilization with CX-3543 (Quarfloxin) selectively enhances cell death in ATRX deficient isogenic cell lines as well as ATRX-mutant primary glioma stem cells derived from patients. Finally, we show that G4 stabilization synergizes with other DNA-damaging therapies, including ionizing radiation, in the ATRX-deficient context. Our findings clarify distinct mechanisms by which DNA secondary structure influences ATRX-deficient glioma pathogenesis and indicate that G4-stabilization may represent and attractive therapeutic strategy for the selective targeting of ATRX-mutant cancers. Opportunities for the development of radiosensitization approaches based on G4-stabilization are particularly intriguing, as ionizing radiation remains among the most effective non-surgical treatments for malignant glioma

Pineoblastoma is a rare and highly aggressive brain cancer of childhood, histologically belonging to the spectrum of primitive neuroectodermal tumors. Patients with germline mutations in DICER1, a ribonuclease involved in microRNA processing, have increased risk of pineoblastoma, but genetic drivers of sporadic pineoblastoma remain unknown. Here, we analyzed pediatric and adult pineoblastoma samples (n = 23) using a combination of genome-wide DNA methylation profiling and whole-exome sequencing or whole-genome sequencing. Pediatric and adult pineoblastomas showed distinct methylation profiles, the latter clustering with lower-grade pineal tumors and normal pineal gland. Recurrent variants were found in genes involved in PKA- and NF-κB signaling, as well as in chromatin remodeling genes. We identified recurrent homozygous deletions of DROSHA, acting upstream of DICER1 in microRNA processing, and a novel microduplication involving chromosomal region 1q21 containing PDE4DIP (myomegalin), comprising the ancient DUF1220 protein domain. Expresion of PDE4DIP and DUF1220 proteins was present exclusively in pineoblastoma with PDE4DIP gain.

BACKGROUND: Pineoblastoma is a rare and highly aggressive brain cancer of childhood, histologically belonging to the spectrum of primitive neuroectodermal tumors. Patients with germline mutations in DICER1, a ribonuclease involved in microRNA processing, have increased risk of pineoblastoma, but genetic drivers of sporadic pineoblastoma remain unknown. METHODS: We analyzed pediatric and adult pineoblastoma samples (n=23) using integrated genomic studies, including genome-wide DNA methylation profiling, whole-exome or whole-genome sequencing, and whole-transcriptome analysis. RESULTS: Pediatric and adult pineoblastomas showed distinct methylation profiles, the latter clustering with lower grade pineal tumors and normal pineal gland. Recurrent somatic mutations were found in genes involved in PKA-and NF-kappaB signaling, as well as in chromatin remodeling genes. We identified recurrent homozygous deletions of DROSHA, acting upstream of DICER1 in microRNA processing, and a novel microduplication involving chromosomal region 1q21 containing PDE4DIP (myomegalin), comprising the ancient DUF1220 protein domain. Expression of PDE4DIP and DUF1220 proteins was present exclusively in pineoblastoma with PDE4DIP gain. Whole-transcriptome analysis showed that homozygous loss of DROSHA led to distinct changes in RNA expression profile. Disruption of the DROSHA locus in human neural stem cells using the CRISPR/Cas9 system, led to decrease of the DROSHA protein, and massive loss of miRNAs. CONCLUSION: We identified recurrent homozygous deletions of DROSHA in pineoblastoma, suggesting that different mechanisms disrupting miRNA processing are involved in the pathogenesis of familial versus sporadic pineoblastoma. Furthermore, a novel microduplication of PDE4DIP leading to upregulation of DUF1220 protein suggests DUF1220 as a novel oncogenic driver in pineoblastoma