Antibody Drug Conjugate Sacituzumab Govitecan Enables A Sequential TOP1/PARP Inhibitor Cancer Therapy Strategy in Breast Cancer Patients.

PURPOSE: The Antibody-Drug Conjugate (ADC) Sacituzumab govitecan (SG) comprises the topoisomerase 1 (TOP1) inhibitor SN-38, coupled to a monoclonal antibody targeting trophoblast cell surface antigen 2 (TROP-2). Poly (ADP-ribose) polymerase (PARP) inhibition may synergize with TOP1 inhibitors and SG, but previous studies combining systemic PARP and TOP1 inhibitors failed due to dose-limiting myelosuppression. Here, we assess proof-of-mechanism and clinical feasibility for SG and talazoparib employing an innovative sequential dosing schedule. PATIENTS AND METHODS: In vitro models tested pharmacodynamic endpoints, and in a phase 1b clinical trial (NCT04039230) 30 patients with metastatic Triple-Negative Breast Cancer (mTNBC) received SG and talazoparib using a concurrent (N=7) or sequential (N=23) schedule. Outcome measures included safety, tolerability, preliminary efficacy and establishment of a recommended phase 2 dose (RP2D). RESULTS: We hypothesized that tumor-selective delivery of TOP1i via SG would reduce non-tumor toxicity and create a temporal window, enabling sequential dosing of SG and PARP inhibition. In vitro, sequential SG followed by talazoparib delayed TOP1 cleavage complex clearance, increased DNA damage and promoted apoptosis. In the clinical trial, sequential SG/talazoparib successfully met primary objectives and demonstrated median PFS of 7.6 months without Dose-Limiting Toxicities (DLTs), while concurrent dosing yielded 2.3 months PFS and multiple DLTs including severe myelosuppression. CONCLUSIONS: While SG dosed concurrently with talazoparib is not tolerated clinically due to an insufficient therapeutic window, sequential dosing of SG then talazoparib proved a viable strategy. These findings support further clinical development of the combination and suggest that ADC-based therapy may facilitate novel, mechanism-based dosing strategies.

Mechanisms of Resistance to Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs), with antibodies targeted against specific antigens linked to cytotoxic payloads, offer the opportunity for a more specific delivery of chemotherapy and other bioactive payloads to minimize side effects. First approved in the setting of HER2+ breast cancer, more recent ADCs have been developed for triple-negative breast cancer (TNBC) and, most recently, hormone receptor-positive (HR+) breast cancer. While antibody-drug conjugates have compared favorably against traditional chemotherapy in some settings, patients eventually progress on these therapies and require a change in treatment. Mechanisms to explain the resistance to ADCs are highly sought after, in hopes of developing next-line treatment options and expanding the therapeutic windows of existing therapies. These resistance mechanisms are categorized as follows: change in antigen expression, change in ADC processing and resistance, and efflux of the ADC payload. This paper reviews the recently published literature on these mechanisms as well as potential options to overcome these barriers.

BRCA1 deficiency in mature CD8+ T lymphocytes impairs antitumor immunity.

Women with BRCA1 germline mutations have approximately an 80% lifetime chance of developing breast cancer. While the tumor suppressor function of BRCA1 in breast epithelium has been studied extensively, it is not clear whether BRCA1 deficiency in non-breast somatic cells also contribute to tumorigenesis. Here, we report that mouse Brca1 knockout (KO) in mature T lymphocytes compromises host antitumor immune response to transplanted syngeneic mouse mammary tumors. T cell adoptive transfer further corroborates CD8+ T cell-intrinsic impact of Brca1 KO on antitumor adaptive immunity. T cell-specific Brca1 KO mice exhibit fewer total CD8+, more exhausted, reduced cytotoxic, and reduced memory tumor-infiltrating T cell populations. Consistent with the preclinical data, cancer-free BRCA1 mutation-carrying women display lower abundance of circulating CD8+ lymphocytes than the age-matched control group. Thus, our findings support the notion that BRCA1 deficiency in adaptive immunity could contribute to BRCA1-related tumorigenesis. We also suggest that prophylactic boosting of adaptive immunity may reduce cancer incidence among at-risk women.

Loss of p53 and genetic evolution in pancreatic cancer: Ordered chaos after the guardian is gone.

A recent Nature study delineates a stepwise genomic evolution during pancreatic cancer development, employing an engineered mutant Kras and heterozygous Trp53 mouse model that identifies cells undergoing Trp53 loss of heterozygosity (LOH). Genetic progression post-Trp53 LOH involves clonal deletions, then genome doubling and subsequent accumulation of subclonal gains and amplifications.

RNA polymerase II pausing factor NELF in CD8+ T cells promotes antitumor immunity.

T cell factor 1 (TCF1) is required for memory and stem-like CD8+ T cell functions. How TCF1 partners with other transcription factors to regulate transcription remains unclear. Here we show that negative elongation factor (NELF), an RNA polymerase II (Pol II) pausing factor, cooperates with TCF1 in T cell responses to cancer. Deletion of mouse Nelfb, which encodes the NELFB subunit, in mature T lymphocytes impairs immune responses to both primary tumor challenge and tumor antigen-mediated vaccination. Nelfb deletion causes more exhausted and reduced memory T cell populations, whereas its ectopic expression boosts antitumor immunity and efficacy of chimeric antigen receptor T-cell immunotherapy. Mechanistically, NELF is associated with TCF1 and recruited preferentially to the enhancers and promoters of TCF1 target genes. Nelfb ablation reduces Pol II pausing and chromatin accessibility at these TCF1-associated loci. Our findings thus suggest an important and rate-limiting function of NELF in anti-tumor immunity.

Differentiation of fetal hematopoietic stem cells requires ARID4B to restrict autocrine KITLG/KIT-Src signaling.

Balance between the hematopoietic stem cell (HSC) duality to either possess self-renewal capacity or differentiate into multipotency progenitors (MPPs) is crucial for maintaining homeostasis of the hematopoietic stem/progenitor cell (HSPC) compartment. To retain the HSC self-renewal activity, KIT, a receptor tyrosine kinase, in HSCs is activated by its cognate ligand KITLG originating from niche cells. Here, we show that AT-rich interaction domain 4B (ARID4B) interferes with KITLG/KIT signaling, consequently allowing HSC differentiation. Conditional Arid4b knockout in mouse hematopoietic cells blocks fetal HSC differentiation, preventing hematopoiesis. Mechanistically, ARID4B-deficient HSCs self-express KITLG and overexpress KIT. As to downstream pathways of KITLG/KIT signaling, inhibition of Src family kinases rescues the HSC differentiation defect elicited by ARID4B loss. In summary, the intrinsic ARID4B-KITLG/KIT-Src axis is an HSPC regulatory program that enables the differentiation state, while KIT stimulation by KITLG from niche cells preserves the HSPC undifferentiated pool.

Tumour DDR1 promotes collagen fibre alignment to instigate immune exclusion.

Immune exclusion predicts poor patient outcomes in multiple malignancies, including triple-negative breast cancer (TNBC)1. The extracellular matrix (ECM) contributes to immune exclusion2. However, strategies to reduce ECM abundance are largely ineffective or generate undesired outcomes3,4. Here we show that discoidin domain receptor 1 (DDR1), a collagen receptor with tyrosine kinase activity5, instigates immune exclusion by promoting collagen fibre alignment. Ablation of Ddr1 in tumours promotes the intratumoral penetration of T cells and obliterates tumour growth in mouse models of TNBC. Supporting this finding, in human TNBC the expression of DDR1 negatively correlates with the intratumoral abundance of anti-tumour T cells. The DDR1 extracellular domain (DDR1-ECD), but not its intracellular kinase domain, is required for immune exclusion. Membrane-untethered DDR1-ECD is sufficient to rescue the growth of Ddr1-knockout tumours in immunocompetent hosts. Mechanistically, the binding of DDR1-ECD to collagen enforces aligned collagen fibres and obstructs immune infiltration. ECD-neutralizing antibodies disrupt collagen fibre alignment, mitigate immune exclusion and inhibit tumour growth in immunocompetent hosts. Together, our findings identify a mechanism for immune exclusion and suggest an immunotherapeutic target for increasing immune accessibility through reconfiguration of the tumour ECM.

Bladder cancer cell-intrinsic PD-L1 signals promote mTOR and autophagy activation that can be inhibited to improve cytotoxic chemotherapy.

Tumor cell-intrinsic programmed death-ligand 1 (PD-L1) signals mediate immunopathologic effects in breast, colon, and ovarian cancers and in melanomas, but bladder cancer (BC) effects are unreported. We show here that BC cell-intrinsic PD-L1 signals in mouse MB49 and human RT4, UM-UC3, and UM-UC-14 BC cells regulate important pathologic pathways and processes, including effects not reported in other cancers. α-PD-L1 antibodies reduced BC cell proliferation in vitro, demonstrating direct signaling effects. BC cell-intrinsic PD-L1 promoted mammalian target of rapamycin complex 1 (mTORC1) signals in vitro and augmented in vivo immune-independent cell growth and metastatic cancer spread, similar to effects we reported in melanoma and ovarian cancer. BC cell-intrinsic PD-L1 signals also promoted basal and stress-induced autophagy, whereas these signals inhibited autophagy in melanoma and ovarian cancer cells. BC cell-intrinsic PD-L1 also mediated chemotherapy resistance to the commonly used BC chemotherapy agents cis-platinum and gemcitabine and to the mTORC1 inhibitor, rapamycin. Thus, BC cell-intrinsic PD-L1 signals regulate important virulence and treatment resistance pathways that suggest novel, actionable treatment targets meriting additional studies. As a proof-of-concept, we showed that the autophagy inhibitor chloroquine improved cis-platinum treatment efficacy in vivo, with greater efficacy in PD-L1 null versus PD-L1-replete BC.

Estrogen receptor beta signaling in CD8+ T cells boosts T cell receptor activation and antitumor immunity through a phosphotyrosine switch.

BackgroundThe non-overlapping functions of the two estrogen receptor subtypes, ERα (Estrogen Receptor α)and ERß (Estrogen Receptor ß), in tumor cells have been studied extensively. However, their counterparts in host cells is vastly underinterrogated. Even less is known about how ERα and ERß activities are regulated in a subtype-specific manner. We previously identified a phosphotyrosine residue (pY36) of human ERß that is important for tumor ERß to inhibit growth of breast cancer cells in vitro and in vivo. A role of this ERß phosphotyrosine switch in regulating host ERß remains unclear.Conventional gene editing was used to mutate the corresponding tyrosine residue of endogenous mouse ERß (Y55F) in mouse embryonic stem cells. The derived homozygous mutant Esr2Y55F/Y55F mouse strain and its wild-type (WT) counterpart were compared in various transplant tumor models for their ability to support tumor growth. In addition, flow cytometry-based immunophenotyping was carried out to assess antitumor immunity of WT and mutant hosts. Adoptive transfer of bone marrow and purified CD8+ T cells were performed to identify the host cell type that harbors ERß-dependent antitumor function. Furthermore, cell signaling assays were conducted to compare T cell receptor (TCR)-initiated signaling cascade in CD8+ T cells of WT and mutant mice. Lastly, the ERß-selective agonist S-equol was evaluated for its efficacy to boost immune checkpoint blockade (ICB)-based anticancer immunotherapy.Disabling the ERß-specific phosphotyrosine switch in tumor-bearing hosts exacerbates tumor growth. Further, a cell-autonomous ERß function was defined in CD8+ effector T cells. Mechanistically, TCR activation triggers ERß phosphorylation, which in turn augments the downstream TCR signaling cascade via a non-genomic action of ERß. S-equol facilitates TCR activation that stimulates the ERß phosphotyrosine switch and boosts anti-PD-1 (Programmed cell death protein 1) ICB immunotherapy.Our mouse genetic study clearly demonstrates a role of the ERß phosphotyrosine switch in regulating ERß-dependent antitumor immunity in CD8+ T cells. Our findings support the development of ERß agonists including S-equol in combination with ICB immunotherapy for cancer treatment.

Genetic ablation of adipocyte PD-L1 reduces tumor growth but accentuates obesity-associated inflammation.

The programmed death-ligand 1 (PD-L1)-dependent immune checkpoint attenuates host immunity and maintains self-tolerance. Imbalance between protective immunity and immunopathology due to altered PD-L1 signaling can lead to autoimmunity or tumor immunosuppression. The role of the PD-L1-dependent checkpoint in non-immune system is less reported. We previously found that white adipocytes highly express PD-L1. Here we show that adipocyte-specific PD-L1 knockout mice exhibit enhanced host anti-tumor immunity against mammary tumors and melanoma with low or no tumor PD-L1. However, adipocyte PD-L1 ablation in tumor-free mice also exacerbates diet-induced body weight gain, pro-inflammatory macrophage infiltration into adipose tissue, and insulin resistance. Low PD-L1 mRNA levels in human adipose tissue correlate with high body mass index and presence of type 2 diabetes. Therefore, our mouse genetic approach unequivocally demonstrates a cell-autonomous function of adipocyte PD-L1 in promoting tumor growth and inhibiting antitumor immunity. In addition, our work uncovers a previously unrecognized role of adipocyte PD-L1 in mitigating obesity-related inflammation and metabolic dysfunction.

PPARγ inhibition boosts efficacy of PD-L1 Checkpoint Blockade Immunotherapy against Murine Melanoma in a sexually dimorphic manner.

Immune checkpoint blockade-based immunotherapy has become standard of care for multiple cancer types. However, the overall response rates among various cancer types still remain unsatisfactory. There is a pressing clinical need to identify combination therapies to improve efficacy of anticancer immunotherapy. We previously showed that pharmacologic inhibition of PPARγ by GW9662 boosts αPD-L1 and αPD-1 antibody efficacy in treating murine mammary tumors. In addition, we defined sexually dimorphic αPD-L1 efficacy in B16 melanoma. Here, we show a sexually dimorphic response to the combination of GW9662 and αPD-L1 immunotherapy in B16 melanoma. Combination effects were observed in female, but not male hosts. Neither female oöphorectomy impairs, nor does male castration rescue the combination effects, suggesting a sex hormone-independent response to this combination therapy. In diet-induced obese females, melanoma growth remained responsive to the combination treatment, albeit less robustly than lean females. These findings are informative for future design and application of immunotherapy-related combination therapy for treating human melanoma patients by taking gender and obesity status into consideration.

Adipose PD-L1 Modulates PD-1/PD-L1 Checkpoint Blockade Immunotherapy Efficacy in Breast Cancer.

Programmed death-ligand 1 (PD-L1) and its receptor programmed cell death protein 1 (PD-1) modulate antitumor immunity and are major targets of checkpoint blockade immunotherapy. However, clinical trials of anti-PD-L1 and anti-PD-1 antibodies in breast cancer demonstrate only modest efficacy. Furthermore, specific PD-L1 contributions in various tissue and cell compartments to antitumor immunity remain incompletely elucidated. Here we show that PD-L1 expression is markedly elevated in mature adipocytes versus preadipocytes. Adipocyte PD-L1 prevents anti-PD-L1 antibody from activating important antitumor functions of CD8+ T cells in vitro. Adipocyte PD-L1 ablation obliterates, whereas forced preadipocyte PD-L1 expression confers, these inhibitory effects. Pharmacologic inhibition of adipogenesis selectively reduces PD-L1 expression in mouse adipose tissue and enhances the antitumor efficacy of anti-PD-L1 or anti-PD-1 antibodies in syngeneic mammary tumor models. Our findings provide a previously unappreciated approach to bolster anticancer immunotherapy efficacy and suggest a mechanism for the role of adipose tissue in breast cancer progression.

The role of focal adhesion kinase in transforming growth factor-ß2 induced migration of human lens epithelial cells.

The migration of lens epithelial cells towards the posterior capsule is a key event in the development of posterior capsule opacification (PCO). Accumulating evidence has described crosstalk between growth factors and adhesive signaling pathways in wound healing and cell migration. The aim of the present study was to elucidate an aberrant transforming growth factor (TGF)­ß2 signaling pathway that regulated the migration of lens epithelial cells in the pathological context of PCO. The expression of fibronectin, focal adhesion kinase (FAK) and phosphorylated (p)­FAK in HLE­B3 cells following TGF­ß2 treatment was determined by western blot analysis and the expression of integrin α5ß1 was detected by flow cytometry. Cell migration capacity was measured by wound healing and Transwell assays in the presence of 1,2,4,5­tetraaminobenzene tetrahydrochloride, a selective FAK inhibitor, fibronectin small interfering RNA interference, arginylglycylaspartic acid peptides or α5ß1­integrin neutralizing antibodies. The 1,2,4,5­tetraaminobenzene tetrahydrochloride was administered daily to 16 rabbits following cataract surgery. Fibronectin and TGF­ß expression were increased in the PCO group, demonstrated by immunofluorescence assays. PCO grading was conducted by slit­lamp biomicroscopy and evaluation of posterior capsule opacification software. It was observed that TGF­ß2 promoted HLE­B3 cell migration and upregulated fibronectin expression, which was followed by an increased phosphorylation of FAK. In addition, TGF­ß2 treatment and fibronectin surface coating significantly increased cell migration and FAK activation, which was inhibited by disrupting fibronectin­integrin α5ß1 interaction with the arginylglycylaspartic acid peptide, α5ß1­integrin neutralizing antibody or fibronectin depletion. Finally, suppression of FAK signaling by its inhibitor significantly decreased cell migration in vitro and attenuated PCO development in vivo. In summary, TGF­ß2 was indicated to promote the migration of lens epithelial cells through the TGF­ß2/fibronectin/integrin/FAK axis. Inhibition of FAK activity decreased TGF­ß2­mediated cell migration in vitro and improved the symptoms of PCO in a rabbit model.

Tumor-extrinsic discoidin domain receptor 1 promotes mammary tumor growth by regulating adipose stromal interleukin 6 production in mice.

Discoidin domain receptor 1 (DDR1) is a collagen receptor that mediates cell communication with the extracellular matrix (ECM). Aberrant expression and activity of DDR1 in tumor cells are known to promote tumor growth. Although elevated DDR1 levels in the stroma of breast tumors are associated with poor patient outcome, a causal role for tumor-extrinsic DDR1 in cancer promotion remains unclear. Here we report that murine mammary tumor cells transplanted to syngeneic recipient mice in which Ddr1 has been knocked out (KO) grow less robustly than in WT mice. We also found that the tumor-associated stroma in Ddr1-KO mice exhibits reduced collagen deposition compared with the WT controls, supporting a role for stromal DDR1 in ECM remodeling of the tumor microenvironment. Furthermore, the stromal-vascular fraction (SVF) of Ddr1 knockout adipose tissue, which contains committed adipose stem/progenitor cells and preadipocytes, was impaired in its ability to stimulate tumor cell migration and invasion. Cytokine array-based screening identified interleukin 6 (IL-6) as a cytokine secreted by the SVF in a DDR1-dependent manner. SVF-produced IL-6 is important for SVF-stimulated tumor cell invasion in vitro, and, using antibody-based neutralization, we show that tumor promotion by IL-6 in vivo requires DDR1. In conclusion, our work demonstrates a previously unrecognized function of DDR1 in promoting tumor growth.

PHBVHHx scaffolds loaded with umbilical cord-derived mesenchymal stem cells or hepatocyte-like cells differentiated from these cells for liver tissue engineering.

More attention has recently been focused on the treatment of various kinds of hepatic diseases based on cell-based therapies. In this study, mesenchymal stem cells were isolated from umbilical cord (UC-MSCs). Results confirmed that UC-MSCs could differentiate into adipocytes, osteoblasts and hepatocytes. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (PHBVHHx), a new member of polyhydroxyalkanoate (PHA) family, was produced by bacteria. Liver-injured mouse model was established by CCl4 injection. PHBVHHx scaffolds were transplanted into the liver-injured mice. Liver morphology on day 28 post-transplantation of scaffolds loaded with UC-MSCs or hepatocyte-like cells differentiated from UC-MSCs significantly improved and looked similar to the normal liver. Concentrations of albumin (ALB) significantly increased, and total bilirubin (TB) and alanine axminotransferase (ALT) significantly decreased on days 14 and 28 post-transplantation of scaffolds loaded with UC-MSCs or differentiated UC-MSCs. HE staining showed that on day 28 post-transplantation of scaffolds loaded with UC-MSCs or differentiated UC-MSCs, livers had similar tissue structure of normal livers. Masson staining showed that on day 28 post-transplantation of scaffolds loaded with UC-MSCs or differentiated UC-MSCs, livers had less blue staining for collagen deposition compared with the others. These results demonstrated that PHBVHHx scaffolds loaded with UC-MSCs or differentiated UC-MSCs had the similar effect on injured livers and significantly promoted the recovery of injured livers.