Chemotherapy-induced differential cell cycle arrest in B-cell lymphomas affects their sensitivity to Wee1 inhibition.

Chemotherapeutic agents, e.g., cytarabine and doxorubicin, cause DNA damage. However, it remains unknown whether such agents differentially regulate cell cycle arrest in distinct types of B-cell lymphomas, and whether this phenotype can be exploited for developing new therapies. We treated various types of B cells, including primary and B lymphoma cells, with cytarabine or doxorubicin, and determined DNA damage responses, cell cycle regulation and sensitivity to a Wee1 inhibitor. We found that cyclin A2/B1 upregulation appears to be an intrinsic programmed response to DNA damage; however, different types of B cells arrest in distinct phases of the cell cycle. The Wee1 inhibitor significantly enhanced the apoptosis of G2 phase-arrested B-cell lymphomas by inducing premature entry into mitosis and mitotic catastrophe, whereas it did not affect G1/S-phase-arrested lymphomas. Cytarabine-induced G1-arrest can be converted to G2-arrest by doxorubicin treatment in certain B-cell lymphomas, which correlates with newly acquired sensitivity to the Wee1 inhibitor. Consequently, the Wee1 inhibitor together with cytarabine or doxorubicin inhibited tumor growth in vitro and in vivo more effectively, providing a potential new therapy for treating B-cell lymphomas. We propose that the differential cell cycle arrest can be exploited to enhance the chemosensitivity of B-cell lymphomas.

Unexpected effects of different genetic backgrounds on identification of genomic rearrangements via whole-genome next generation sequencing.

BACKGROUND: Whole genome next generation sequencing (NGS) is increasingly employed to detect genomic rearrangements in cancer genomes, especially in lymphoid malignancies. We recently established a unique mouse model by specifically deleting a key non-homologous end-joining DNA repair gene, Xrcc4, and a cell cycle checkpoint gene, Trp53, in germinal center B cells. This mouse model spontaneously develops mature B cell lymphomas (termed G1XP lymphomas). RESULTS: Here, we attempt to employ whole genome NGS to identify novel structural rearrangements, in particular inter-chromosomal translocations (CTXs), in these G1XP lymphomas. We sequenced six lymphoma samples, aligned our NGS data with mouse reference genome (in C57BL/6J (B6) background) and identified CTXs using CREST algorithm. Surprisingly, we detected widespread CTXs in both lymphomas and wildtype control samples, majority of which were false positive and attributable to different genetic backgrounds. In addition, we validated our NGS pipeline by sequencing multiple control samples from distinct tissues of different genetic backgrounds of mouse (B6 vs non-B6). Lastly, our studies showed that widespread false positive CTXs can be generated by simply aligning sequences from different genetic backgrounds of mouse. CONCLUSIONS: We conclude that mapping and alignment with reference genome might not be a preferred method for analyzing whole-genome NGS data obtained from a genetic background different from reference genome. Given the complex genetic background of different mouse strains or the heterogeneity of cancer genomes in human patients, in order to minimize such systematic artifacts and uncover novel CTXs, a preferred method might be de novo assembly of personalized normal control genome and cancer cell genome, instead of mapping and aligning NGS data to mouse or human reference genome. Thus, our studies have critical impact on the manner of data analysis for cancer genomics.

PD1+TIGIT+2B4+KLRG1+ Cells Might Underlie T Cell Dysfunction in Patients Treated with BCMA-Directed Chimeric Antigen Receptor T Cell Therapy.

Chimeric antigen receptor T cell (CAR-T) therapy has shown rapid, frequent, and deep responses in patients with relapsed/refractory multiple myeloma (RRMM). However, relapse frequently occurs following CAR-T therapy, and the cause of this resistance is not well defined. Among the potential mechanisms of resistance, T cell intrinsic factors may be an important source of failure. Here we used spectral flow cytometry to identify the changes in T cell phenotypes in bone marrow aspirates at different stages of multiple myeloma progression, including cases that relapsed after anti-BCMA CAR-T therapy. We identified completely different T cell phenotypes in RRMM and post CAR-T relapse cases compared to healthy donors and earlier stages of multiple myeloma, novel double-negative CD3+ T cells in RRMM and CAR-T relapsed cases, and differences in CD8 T cell phenotype at the baseline between peripheral blood and bone marrow from healthy donors. We found that the majority of T cells in RRMM patients and significant T cell subsets in post-CAR-T relapsed patients expressed multiple coinhibitory markers, including PD1, TIGIT, 2B4, and KLRG1.

Preclinical development of CD126 CAR-T cells with broad antitumor activity.

Chimeric antigen receptor T (CAR-T) cell therapy is a transformative approach to cancer eradication. CAR-T is expensive partly due to the restricted use of each CAR construct for specific tumors. Thus, a CAR construct with broad antitumor activity can be advantageous. We identified that CD126 is expressed by many hematologic and solid tumors, including multiple myeloma, lymphoma, acute myeloid leukemia, pancreatic and prostate adenocarcinoma, non-small cell lung cancer, and malignant melanoma among others. CAR-T cells targeting CD126 were generated and shown to kill many tumor cells in an antigen-specific manner and with efficiency directly proportional to CD126 expression. Soluble CD126 did not interfere with CAR-T cell killing. The CAR-T constructs bind murine CD126 but caused no weight loss or hepatotoxicity in mice. In multiple myeloma and prostate adenocarcinoma xenograft models, intravenously injected CD126 CAR-T cells infiltrated within, expanded, and killed tumor cells without toxicity. Binding of soluble interleukin-6 receptor (sIL-6R) by CAR-T cells could mitigate cytokine release syndrome. Murine SAA-3 levels were lower in mice injected with CD126 CAR-T compared to controls, suggesting that binding of sIL-6R by CAR-T cells could mitigate cytokine release syndrome. CD126 provides a novel therapeutic target for CAR-T cells for many tumors with a low risk of toxicity.

Metformin inhibits IL-6 signaling by decreasing IL-6R expression on multiple myeloma cells.

IL-6 signaling plays a crucial role in the pathogenesis of a number of diseases, including multiple myeloma, primary amyloidosis, cytokine release syndrome and other inflammatory conditions. It is central for the growth and survival of malignant plasma cells. IL-6R and IL-6ST receptors transduce IL-6 signaling. Molecular mechanisms regulating expression of IL-6R are not well understood and current therapies are based on monoclonal antibody to target IL-6 signaling. Small molecule inhibitors targeting IL-6 signaling are highly desirable. Metformin specifically decreased IL-6R expression which is mediated via AMPK, mTOR, and miR34a. This is a novel finding and adds to existing therapies targeting IL-6 signaling.

Leptin signals via TGFB1 to promote metastatic potential and stemness in breast cancer.

Epidemiological studies have shown obesity to be linked with poorer outcomes in breast cancer patients. The molecular mechanisms responsible for the increased risk of invasive/metastatic disease with obesity are complex, but may include elevated levels of adipokines such as leptin. Using physiological levels of leptin found in obesity in a novel chronic in vitro treatment model (≤200 ng/ml for 14 days), we confirmed the occurrence of leptin-mediated changes in growth, apoptosis and metastatic behavior, and gene expression changes representing epithelial-to-mesenchymal transition (EMT) and a cancer stem cell (CSC) like phenotype in breast epithelial and cancer cell lines (MCF10A, MCF10AT1, MCF7 and MDA-MB-231). Further, we have discovered that these effects were accompanied by increased expression of TGFB1, and could be significantly reduced by co-treatment with neutralizing antibody against TGFB1, indicating that the induction of these characteristics was mediated via TGFB1. Occurring in both MCF7 and MCF10AT1 cells, it suggests these actions of leptin to be independent of estrogen receptor status. By linking leptin signalling to the established TGFB1 pathway of metastasis / EMT, this study gives a direct mechanism by which leptin can contribute to the poorer outcomes of obese cancer patients. Inhibitors of TGFB1 are in currently in phase III clinical trials in other malignancies, thus identifying the connection between leptin and TGFB1 will open new therapeutic opportunities for improving outcomes for obese breast cancer patients.

Fulvestrant inhibits growth of triple negative breast cancer and synergizes with tamoxifen in ERα positive breast cancer by up-regulation of ERß.

The estrogen receptor-alpha (ERα) is used as a predictive marker for anti-estrogen therapy in breast cancer patients. In addition to aromatase inhibitors, ERα can be targeted at the receptor level using the receptor modulator tamoxifen or by the pure anti-estrogen fulvestrant. The role of the second ER, ER-beta (ERß), as a therapeutic target or prognostic marker in breast cancer is still elusive. Hitherto, it is not known if ERα+/ERß+ breast cancers would benefit from a treatment strategy combining tamoxifen and fulvestrant or if fulvestrant exert any therapeutic effects in ERα-/ERß+ breast cancer. Here, we report that fulvestrant up-regulated ERß in ERα+/ERß+ breast cancer and in triple negative ERß+ breast cancers (ERα-/ERß+). In ERα+/ERß+ breast cancer, a combination therapy of tamoxifen and fulvestrant significantly reduced tumor growth compared to either treatment alone both in vivo and in vitro. In ERα-/ERß+ breast cancer fulvestrant had potent effects on cancer growth, in vivo as well as in vitro, and this effect was dependent on intrinsically expressed levels of ERß. The role of ERß was further confirmed in cells where ERß was knocked-in or knocked-down. Inhibition of DNA methyltransferase (DNMT) increased the levels of ERß and fulvestrant exerted similar potency on DNMT activity as the DNMT inhibitor decitabine. We conclude that fulvestrant may have therapeutic potential in additional groups of breast cancer patients; i) in ERα+/ERß+ breast cancer where fulvestrant synergizes with tamoxifen and ii) in triple negative/ERß+ breast cancer patients, a subgroup of breast cancer patients with poor prognosis.

Squamous cell carcinomas escape immune surveillance via inducing chronic activation and exhaustion of CD8+ T Cells co-expressing PD-1 and LAG-3 inhibitory receptors.

Squamous cell carcinoma (SCC) is the second commonest type of skin cancer. Moreover, about 90% of head and neck cancers are SCCs. SCCs develop at a significantly higher rate under chronic immunosuppressive conditions, implicating a role of immune surveillance in controlling SCCs. It remains largely unknown how SCCs evade immune recognition. Here, we established a mouse model by injecting tumor cells derived from primary SCCs harboring KrasG12D mutation and Smad4 deletion into wild-type (wt) or CD8-/- recipients. We found comparable tumor growth between wt and CD8-/- recipients, indicating a complete escape of CD8+ T cell-mediated anti-tumor responses by these SCCs. Mechanistically, CD8+ T cells apparently were not defective in infiltrating tumors given their relatively increased percentage among tumor infiltrating lymphocytes (TILs). CD8+ TILs exhibited phenotypes of chronic activation and exhaustion, including overexpression of activation markers, co-expression of programmed cell death 1 (PD-1) and lymphocyte activation gene-3 (LAG-3), as well as TCRß downregulation. Among CD4+ TILs, T regulatory cells (Tregs) were preferentially expanded. Contradictory to prior findings in melanoma, Treg expansion was independent of CD8+ T cells in our SCC model. Unexpectedly, CD8+ T cells were required for promoting NK cell infiltration within SCCs. Furthermore, we uncovered AKT-dependent lymphocyte-induced PD-L1 upregulation on SCCs, which was contributed greatly by combinatorial effects of CD8+ T and NK cells. Lastly, dual blockade of PD-1 and LAG-3 inhibited the tumor growth of SCCs. Thus, our findings identify novel immune evasion mechanisms of SCCs and suggest that immunosuppressive mechanisms operate in a cancer-type specific and context-dependent manner.