CDK inhibition results in pharmacologic BRCAness increasing sensitivity to olaparib in BRCA1-WT and olaparib resistant in Triple Negative Breast Cancer.

One in three Triple Negative Breast Cancer (TNBC) is Homologous Recombination Deficient (HRD) and susceptible to respond to PARP inhibitor (PARPi), however, resistance resulting from functional HR restoration is frequent. Thus, pharmacologic approaches that induce HRD are of interest. We investigated the effectiveness of CDK-inhibition to induce HRD and increase PARPi sensitivity of TNBC cell lines and PDX models. Two CDK-inhibitors (CDKi), the broad range dinaciclib and the CDK12-specific SR-4835, strongly reduced the expression of key HR genes and impaired HR functionality, as illustrated by BRCA1 and RAD51 nuclear foci obliteration. Consequently, both CDKis showed synergism with olaparib, as well as with cisplatin and gemcitabine, in a range of TNBC cell lines and particularly in olaparib-resistant models. In vivo assays on PDX validated the efficacy of dinaciclib which increased the sensitivity to olaparib of 5/6 models, including two olaparib-resistant and one BRCA1-WT model. However, no olaparib response improvement was observed in vivo with SR-4835. These data support that the implementation of CDK-inhibitors could be effective to sensitize TNBC to olaparib as well as possibly to cisplatin or gemcitabine.

A novel Fc-engineered cathepsin D-targeting antibody enhances ADCC, triggers tumor-infiltrating NK cell recruitment, and improves treatment with paclitaxel and enzalutamide in triple-negative breast cancer.

INTRODUCTION: Triple-negative breast cancer (TNBC) prognosis is poor. Immunotherapies to enhance the antibody-induced natural killer (NK) cell antitumor activity are emerging for TNBC that is frequently immunogenic. The aspartic protease cathepsin D (cath-D), a tumor cell-associated extracellular protein with protumor activity and a poor prognosis marker in TNBC, is a prime target for antibody-based therapy to induce NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). This study investigated whether Fc-engineered anti-cath-D antibodies trigger ADCC, their impact on antitumor efficacy and tumor-infiltrating NK cells, and their relevance for combinatory therapy in TNBC. METHODS: Cath-D expression and localization in TNBC samples were evaluated by western blotting, immunofluorescence, and immunohistochemistry. The binding of human anti-cath-D F1M1 and Fc-engineered antibody variants, which enhance (F1M1-Fc+) or prevent (F1M1-Fc-) affinity for CD16a, to secreted human and murine cath-D was analyzed by ELISA, and to CD16a by surface plasmon resonance and flow cytometry. NK cell activation was investigated by flow cytometry, and ADCC by lactate dehydrogenase release. The antitumor efficacy of F1M1 Fc-variants was investigated using TNBC cell xenografts in nude mice. NK cell recruitment, activation, and cytotoxic activity were analyzed in MDA-MB-231 cell xenografts by immunophenotyping and RT-qPCR. NK cells were depleted using an anti-asialo GM1 antibody. F1M1-Fc+ antitumor effect was assessed in TNBC patient-derived xenografts (PDXs) and TNBC SUM159 cell xenografts, and in combination with paclitaxel or enzalutamide. RESULTS: Cath-D expression on the TNBC cell surface could be exploited to induce ADCC. F1M1 Fc-variants recognized human and mouse cath-D. F1M1-Fc+ activated NK cells in vitro and induced ADCC against TNBC cells and cancer-associated fibroblasts more efficiently than F1M1. F1M1-Fc- was ineffective. In the MDA-MB-231 cell xenograft model, F1M1-Fc+ displayed higher antitumor activity than F1M1, whereas F1M1-Fc- was less effective, reflecting the importance of Fc-dependent mechanisms in vivo. F1M1-Fc+ triggered tumor-infiltrating NK cell recruitment, activation and cytotoxic activity in MDA-MB-231 cell xenografts. NK cell depletion impaired F1M1-Fc+ antitumor activity, demonstrating their key role. F1M1-Fc+ inhibited growth of SUM159 cell xenografts and two TNBC PDXs. In combination therapy, F1M1-Fc+ improved paclitaxel and enzalutamide therapeutic efficacy without toxicity. CONCLUSIONS: F1M1-Fc+ is a promising immunotherapy for TNBC that could be combined with conventional regimens, including chemotherapy or antiandrogens.

BRCA1-methylated triple negative breast cancers previously exposed to neoadjuvant chemotherapy form RAD51 foci and respond poorly to olaparib.

Background: About 15% of Triple-Negative-Breast-Cancer (TNBC) present silencing of the BRCA1 promoter methylation and are assumed to be Homologous Recombination Deficient (HRD). BRCA1-methylated (BRCA1-Me) TNBC could, thus, be eligible to treatment based on PARP-inhibitors or Platinum salts. However, their actual HRD status is discussed, as these tumors are suspected to develop resistance after chemotherapy exposure. Methods: We interrogated the sensitivity to olaparib vs. carboplatin of 8 TNBC Patient-Derived Xenografts (PDX) models. Four PDX corresponded to BRCA1-Me, of which 3 were previously exposed to NeoAdjuvant-Chemotherapy (NACT). The remaining PDX models corresponded to two BRCA1-mutated (BRCA1-Mut) and two BRCA1-wild type PDX that were respectively included as positive and negative controls. The HRD status of our PDX models was assessed using both genomic signatures and the functional BRCA1 and RAD51 nuclear foci formation assay. To assess HR restoration associated with olaparib resistance, we studied pairs of BRCA1 deficient cell lines and their resistant subclones. Results: The 3 BRCA1-Me PDX that had been exposed to NACT responded poorly to olaparib, likewise BRCA1-WT PDX. Contrastingly, 3 treatment-naïve BRCA1-deficient PDX (1 BRCA1-Me and 2 BRCA1-mutated) responded to olaparib. Noticeably, the three olaparib-responsive PDX scored negative for BRCA1- and RAD51-foci, whereas all non-responsive PDX models, including the 3 NACT-exposed BRCA1-Me PDX, scored positive for RAD51-foci. This suggested HRD in olaparib responsive PDX, while non-responsive models were HR proficient. These results were consistent with observations in cell lines showing a significant increase of RAD51-foci in olaparib-resistant subclones compared with sensitive parental cells, suggesting HR restoration in these models. Conclusion: Our results thus support the notion that the actual HRD status of BRCA1-Me TNBC, especially if previously exposed to chemotherapy, may be questioned and should be verified using the BRCA1- and RAD51-foci assay.