Solid cancer-directed CAR T cell therapy that attacks both tumor and immunosuppressive cells via targeting PD-L1.

Chimeric antigen receptor (CAR) T cell therapy has encountered limited success in solid tumors. The lack of dependable antigens and the immunosuppressive tumor microenvironment (TME) are major challenges. Within the TME, tumor cells along with immunosuppressive cells employ an immune-evasion mechanism that upregulates programmed death ligand 1 (PD-L1) to deactivate effector T cells; this makes PD-L1 a reliable, universal target for solid tumors. We developed a novel PD-L1 CAR (MC9999) using our humanized anti-PD-L1 monoclonal antibody, designed to simultaneously target tumor and immunosuppressive cells. The antigen-specific antitumor effects of MC9999 CAR T cells were observed consistently across four solid tumor models: breast cancer, lung cancer, melanoma, and glioblastoma multiforme (GBM). Notably, intravenous administration of MC9999 CAR T cells eradicated intracranially established LN229 GBM tumors, suggesting penetration of the blood-brain barrier. The proof-of-concept data demonstrate the cytolytic effect of MC9999 CAR T cells against immunosuppressive cells, including microglia HMC3 cells and M2 macrophages. Furthermore, MC9999 CAR T cells elicited cytotoxicity against primary tumor-associated macrophages within GBM tumors. The concept of targeting both tumor and immunosuppressive cells with MC9999 was further validated using CAR T cells derived from cancer patients. These findings establish MC9999 as a foundation for the development of effective CAR T cell therapies against solid tumors.

Molecular profiling reveals a hypoxia signature in breast implant-associated anaplastic large cell lymphoma.

Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a recently characterized T-cell malignancy that has raised significant patient safety concerns and led to worldwide impact on the implants used and clinical management of patients undergoing reconstructive or cosmetic breast surgery. Molecular signatures distinguishing BIA-ALCL from other ALCLs have not been fully elucidated and classification of BIA-ALCL as a WHO entity remains provisional. We performed RNA sequencing and gene set enrichment analysis comparing BIA-ALCLs to non-BIA-ALCLs and identified dramatic upregulation of hypoxia signaling genes including the hypoxia-associated biomarker CA9 (carbonic anyhydrase-9). Immunohistochemistry validated CA9 expression in all BIA-ALCLs, with only minimal expression in non-BIA-ALCLs. Growth induction in BIA-ALCL-derived cell lines cultured under hypoxic conditions was proportional to up-regulation of CA9 expression, and RNA sequencing demonstrated induction of the same gene signature observed in BIA-ALCL tissue samples compared to non-BIA-ALCLs. CA9 silencing blocked hypoxia-induced BIA-ALCL cell growth and cell cycle-associated gene expression, whereas CA9 overexpression in BIA-ALCL cells promoted growth in a xenograft mouse model. Furthermore, CA9 was secreted into BIA-ALCL cell line supernatants and was markedly elevated in human BIA-ALCL seroma samples. Finally, serum CA9 concentrations in mice bearing BIA-ALCL xenografts were significantly elevated compared to control serum. Together, these findings characterize BIA-ALCL as a hypoxia-associated neoplasm, likely attributable to the unique microenvironment in which it arises. These data support classification of BIA-ALCL as a distinct entity and uncover opportunities for investigating hypoxia-related proteins such as CA9 as novel biomarkers and therapeutic targets in this disease.

Identifying Synthetic Lectins from a Competitive Screen for the Detection of Prostate Cancer.

Herein, we report a dual dye competitive screening method for the identification of five boronic acid functionalized synthetic lectins (SLs) that are selective for prostate-associated targets with the goal of detecting and staging prostate cancer. This method uses differently labeled normal (RWEP-1) and diseased (PC3) cell membrane extracts in a competitive binding assay to identify SLs that bind either the cancerous or normal extracts but not both. Subsequent studies examined the efficacy of these new SL hits in an array format to discriminate six prostate cell lines. The SL array was able to (a) classify the prostate cell lines with 83% accuracy, (b) discriminate the same cell lines based on their metastatic potential (noncancerous/healthy, cancerous/lowly metastatic, and cancerous/metastatic) with 96% classification accuracy, and (c) exhibit enhanced selectivity for prostate-derived versus colon-derived cell lines. Further analysis delineated the contribution from each SL in these studies, providing a focused SL array having potential utility as a cancer diagnostic.

Recurrent MSC E116K mutations in ALK-negative anaplastic large cell lymphoma.

Anaplastic large cell lymphomas (ALCLs) represent a relatively common group of T-cell non-Hodgkin lymphomas (T-NHLs) that are unified by similar pathologic features but demonstrate marked genetic heterogeneity. ALCLs are broadly classified as being anaplastic lymphoma kinase (ALK)+ or ALK-, based on the presence or absence of ALK rearrangements. Exome sequencing of 62 T-NHLs identified a previously unreported recurrent mutation in the musculin gene, MSC E116K, exclusively in ALK- ALCLs. Additional sequencing for a total of 238 T-NHLs confirmed the specificity of MSC E116K for ALK- ALCL and further demonstrated that 14 of 15 mutated cases (93%) had coexisting DUSP22 rearrangements. Musculin is a basic helix-loop-helix (bHLH) transcription factor that heterodimerizes with other bHLH proteins to regulate lymphocyte development. The E116K mutation localized to the DNA binding domain of musculin and permitted formation of musculin-bHLH heterodimers but prevented their binding to authentic target sequence. Functional analysis showed MSCE116K acted in a dominant-negative fashion, reversing wild-type musculin-induced repression of MYC and cell cycle inhibition. Chromatin immunoprecipitation-sequencing and transcriptome analysis identified the cell cycle regulatory gene E2F2 as a direct transcriptional target of musculin. MSCE116K reversed E2F2-induced cell cycle arrest and promoted expression of the CD30-IRF4-MYC axis, whereas its expression was reciprocally induced by binding of IRF4 to the MSC promoter. Finally, ALCL cells expressing MSC E116K were preferentially targeted by the BET inhibitor JQ1. These findings identify a novel recurrent MSC mutation as a key driver of the CD30-IRF4-MYC axis and cell cycle progression in a unique subset of ALCLs.