Emergence of immunotherapies for treating cancer has been an important step towards a more efficient, selective and safe approach for patients. However, only a fraction of patients can respond to treatments currently available, requiring the identification of additional therapeutic targets. The relevance of preclinical models in the identification of new therapeutic targets and assessment of immunotherapies has vastly improved with mice bearing a human immune system. BRGSF (Balb/C Rag2-/-, IL2Rγ-/-, SIRPαNOD and Flt3-/-) is a highly immunodeficient mouse featuring reduced murine myeloid cells. BRGSF mice reconstituted with human cord blood CD34+ cells (BRGSF-HIS) develop functional lymphoid and myeloid compartments. This engraftment is stable for over a year (Labarthe et al., 2019) and mice do not develop GvHD. Human myeloid compartment can be transiently boosted with exogenous human Flt3L injections. In contrary to other models, which overexpress human cytokines to develop human myeloid cells, Flt3L-treated BRGSF-HIS mice do not show side effects. BRGSF-HIS mice are permissive to mouse and human cancer cell lines engraftment and represent a valuable preclinical model to study cancer development and evaluate novel therapeutics. Here, we show that composition of tumor microenvironment (TME) is tumor burden-dependent. Indeed, BRGSF-HIS mice engrafted with triple negative cancer cell line MDA-MB-231 have a diverse TME, enriched in myeloid cells. The major cell type present in tumors of approximately 200mm3 is CD206+/CD163+ M2-like macrophages, which express high levels of PD-L1 and other immunomodulatory proteins. At this tumor volume, TME is also composed of a small fraction of T cells. However, the frequency of conventional CD4+T cells increase from <5% to 15% of human infiltrate when tumors reach approximately 500 mm3. Interestingly, NK cells are detected at very low levels in tumors of 200 mm3, but increase to 10% in the TME of tumors of 500 mm3. The infiltration of human immune cells in the TME is also tumor type-dependent, as in a colon-derived adenocarcinoma SW480 tumor model, conventional CD4+ T cells are the major cell type present in the TME (40%) of tumors of 200 mm3. Interestingly, these tumors show an infiltrate of γδ T cells as well, ranging from 6% to 8%of human immune cells in tumors of 200 mm3 and 800 mm3, respectively. BRGSF-HIS mice hence represent a valuable tool to investigate immune cell infiltration in the TME, enabling a translatable assessment of mechanism of action of immunotherapies. Florent Creusat, Siham Hedir, Alexis Gonon, Amélie Marguier, Yacine Cherifi, Fabiane Sonego, Gaëlle Martin, Kader Thiam. Tumor microenvironment composition is shaped by tumor cell line-derived xenograft subtype and tumor burden in BRGSF-HIS mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3126.

Abstract The relevance of preclinical models has vastly improved with mice bearing a human immune system, especially in the context of immunotherapy. BRGSF (Balb/C Rag2−/−, IL2Rγ−/−, SIRPαNOD and Flt3−/−) is a highly immunodeficient mouse featuring reduced murine myeloid cells. BRGSF mice reconstituted with human cord blood CD34+ cells (BRGSF-HIS) develop functional lymphoid and myeloid compartments. This engraftment is stable for over a year (Labarthe et al., 2019) and mice do not develop GvHD. Myeloid compartment can be transiently boosted with exogenous human Flt3L injections. In contrary to other models overexpressing human cytokines to develop human myeloid cells, Flt3L-treated BRGSF-HIS mice do not show side effects. BRGSF-HIS mice are permissive to mouse and human cancer cell lines engraftment and represent a valuable preclinical model to study cancer development and test novel therapeutics. Effect of Flt3L-induced boost on myeloid compartment is seen in a non-small cell lung cancer (A549) tumor model. A single boost prior to tumor cell inoculation associates with increased tumor-infiltrating T-cells (mainly CD8+ T-cells) and myeloid cells, and reduced tumor-infiltrating NK cells in the tumor microenvironment (TME). Human triple negative cancer cell (TNBC) MDA-MB-231 and human pancreatic adenocarcinoma HPAFII cell lines are widely used in cancer research and drug development. We show that upon implantation in BRGSF-HIS mice, in vivo growth of human cell line-derived xenograft (CDX) is CD34+ donor independent and the TME composition varies according to cell line used. As seen in TNBC patients (Zheng et al., 2021); TME of MDA-MB-231 bearing BRGSF-HIS mice is enriched in myeloid cells, mostly CD206+/CD163+ M2 macrophages. CD163-expressing M2 macrophages represent the main tumor-associated macrophages, and are known to promote breast cancer initiation, angiogenesis, invasion, and metastasis by generating an immunosuppressive TME. Interestingly, TME composition evolves over time, with increased T cells frequency in later stage (tumor volume over 1000 mm3 compared to 500 mm3) depicting cancer cell plasticity. Conversely, TME of HPAFII bearing mice is mainly composed of T cells enabling T cells-based therapy. Treatment of BRGSF-HIS mice bearing HPAFII tumor cells with combotherapy based on CD3xTAA + CD28xTAA efficiently reduces tumor growth in vivo compared to vehicle-treated mice or mice injected with CD3xTAA alone. Induced systemic immunomodulation is observed in the TME where increased numbers of both CD4 and CD8 T cells are observed upon combotherapy. Remarkably, while no CD34+ donor effect is observed in CDX growth kinetic, response to treatment appears to be donor dependent with apparition of “weak” and “good” responders. This heterogeneity of response mimics what is observed in clinic and enables further investigations of treatment mode of action and immune escape mechanisms. Citation Format: Perrine Martin-Jeantet, Siham Hedir, Fabiane Sonego, Gaëlle Martin, Yacine Cherifi, Kader Thiam. Tumor cell line-derived xenograft subtype shapes tumor microenvironment composition in BRGSF-HIS mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1530.

In the area of cancer research, the development of new and potent inhibitors of anti-apoptotic proteins is a very active and promising topic. The small molecule MIM1 has been reported earlier as one of the first selective inhibitors of the anti-apoptotic protein Mcl-1. In the present paper, we first revised the structure of this molecule based on extensive physicochemical analyses. Then we designed and synthesized a focused library of analogues for the corrected structure of MIM1. Next, these molecules were subjected to a panel of in cellulo biological studies, allowing the identification of dual Bcl-xL/Mcl-1 inhibitors, as well as selective Mcl-1 inhibitors. These results have been complemented by fluorescence polarization assays with the Mcl-1 protein. Preliminary structure-activity relationships were discussed and extensive molecular modelling studies allowed us to propose a rationale for the biological activity of this series of new inhibitors, in particular for the selectivity of inhibition of Mcl-1 versus Bcl-xL.

Protein-protein interactions are attractive targets because they control numerous cellular processes. In oncology, apoptosis regulating Bcl-2 family proteins are of particular interest. Apoptotic cell death is controlled via PPIs between the anti-apoptotic proteins hydrophobic groove and the pro-apoptotic proteins BH3 domain. In ovarian carcinoma, it has been previously demonstrated that Bcl-xL and Mcl-1 cooperate to protect tumor cells against apoptosis. Moreover, Mcl-1 is a key regulator of cancer cell survival and is a known resistance factor to Bcl-2/Bcl-xL pharmacological inhibitors making it an attractive therapeutic target. Here, using a structure-guided design from the oligopyridine lead Pyridoclax based on Noxa/Mcl-1 interaction we identified a new derivative, active at lower concentration as compared to Pyridoclax. This new derivative selectively binds to the Mcl-1 hydrophobic groove and releases Bak and Bim from Mcl-1 to induce cell death and sensitize cancer cells to Bcl-2/Bcl-xL targeting strategies.

Abstract Ovarian cancer is the leading cause of death from gynecological malignancies worldwide. Although the patients are initially quite sensitive to the taxane and platinum-based first-line chemotherapy, most of them relapse and develop chemoresistance. Defects in apoptosis regulation in ovarian cancer allow the cancer cells to evade cell death and contribute to chemoresistance. Mcl-1 is an anti-apoptotic member of the Bcl-2 proteins family and its amplifıcation is one of the most frequent genetic aberrations found in human cancers. Its expression is at the origin of the acquired resistance to chemotherapy and to Bcl-2 and Bcl-xL inhibitors. In ovarian cancers, we previously demonstrated that Bcl-xL and Mcl-1 cooperate to prevent cancer cells from undergoing apoptotic cell death. Their concomitant inhibitions lead to massive apoptosis even in absence of chemotherapy. Moreover, in some cases, Mcl-1 inhibition is itself able to lead to apoptosis. If clinically relevant pharmacologic inhibition of Bcl-xL is available using ABT-263, selective direct inhibition of Mcl-1 remains problematic. In this context, our teams have designed and synthesized small compounds based on a pyridyl scaffold, named oligopyridines, which potentially target the Mcl-1 hydrophobic binding pocket. We demonstrated that the lead of the first generation of oligopyridines, named Pyridoclax, interacts directly with Mcl-1, releases its pro-apoptotic partners Bim and Bak and induces massive apoptosis at 25 µM concentration in combination with anti-Bcl-xL strategies in chemoresistant ovarian cancer cell lines (Gloaguen et al., J Med Chem 2015). In the present study, we investigated the antitumor activity of Pyridoclax hydrochloride in three subcutaneous xenograft models derived from the injection of chemoresistant ovarian cancer cell lines. Different routes of Pyridoclax hydrochloride administration were tested and its antitumor effect was analyzed at different doses as single agent or in combination with ABT-263. This study highlighted an effective antitumor activity of 20mg/kg of Pyridoclax administered intravenously as single agent in two of three xenograft models without side effects. In order to improve its biological activity, we evaluated the cytotoxic effects of a second generation of oligopyridines derived from the Pyridoclax. This allowed us to identify the MR31367, one of the most potent oligopyridines that shows a stronger pro-apoptotic activity in association with to Bcl-xL-targeting strategies in ovarian cancer cell lines. Further characterization showed that this derivative binds Mcl-1 and release Bim and Bak from it, leading to Bak-mediated apoptosis. Overall, these results open up interesting perspectives for the clinical use of Mcl-1 inhibitors as single agent or in combination with anticancer drugs to improve the clinical management of ovarian cancers. Citation Format: Siham Hedir, Louis-Bastien Weiswald, Marcella De Giorgi, Jade Fogha, Martina De Pascale, Emilie Brotin, Bogdan Marekha, Peggy Suzanne, Fabien Gautier, Philippe Juin, Laetitia Ligat, Frédéric Lopez, Rémi Legay, Ronan Bureau, Sylvain Rault, Jana Sopkova-de Oliveira Santos, Anne-Sophie Voisin-Chiret, Laurent Poulain. Pyridoclax and its derivatives from oligopyridine family directly inhibit Mcl-1 and exert potent antitumor effects on ovarian cancer in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3996.

Ovarian cancer is the leading cause of death from gynecological malignancies worldwide, and innate or acquired chemoresistance of ovarian cancer cells is the major cause of therapeutic failure. It has been demonstrated that the concomitant inhibition of Bcl-xL and Mcl-1 anti-apoptotic activities is able to trigger apoptosis in chemoresistant ovarian cancer cells. In this context, siRNA-mediated Bcl-xL and Mcl-1 inhibition constitutes an appealing strategy by which to eliminate chemoresistant cancer cells. However, the safest and most efficient way to vectorize siRNAs in vivo is still under debate. In the present study, using in vivo bioluminescence imaging, we evaluated the interest of atelocollagen to vectorize siRNAs by intraperitoneal (i.p.) or intravenous (i.v.) administration in 2 xenografted ovarian cancer models (peritoneal carcinomatosis and subcutaneous tumors in nude mice). Whereas i.p. administration of atelocollagen-vectorized siRNA in the peritoneal carcinomatosis model did not induce any gene downregulation, a 70% transient downregulation of luciferase expression was achieved after i.v. injection of atelocollagen-vectorized siRNA in the subcutaneous (s.c.) model. However, the use of siRNA targeting Bcl-xL or Mcl-1 did not induce target-specific downregulation in vivo in nude mice. Our results therefore show that atelocollagen complex formulation, the administration route, tumor site and the identity of the siRNA target influence the efficiency of atelocollagen-mediated siRNA delivery.