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Purpose: The identification of a molecular signature predicting the relapse of tamoxifen-treated primary breast cancers should help the therapeutic management of estrogen receptor–positive cancers.Experimental Design: A series of 132 primary tumors from patients who received adjuvant tamoxifen were analyzed for expression profiles at the whole-genome level by 70-mer oligonucleotide microarrays. A supervised analysis was done to identify an expression signature.Results: We defined a 36-gene signature that correctly classified 78% of patients with relapse and 80% of relapse-free patients (79% accuracy). Using 23 independent tumors, we confirmed the accuracy of the signature (78%) whose relevance was further shown by using published microarray data from 60 tamoxifen-treated patients (63% accuracy). Univariate analysis using the validation set of 83 tumors showed that the 36-gene classifier is more efficient in predicting disease-free survival than the traditional histopathologic prognostic factors and is as effective as the Nottingham Prognostic Index or the “Adjuvant!” software. Multivariate analysis showed that the molecular signature is the only independent prognostic factor. A comparison with several already published signatures demonstrated that the 36-gene signature is among the best to classify tumors from both training and validation sets. Kaplan-Meier analyses emphasized its prognostic power both on the whole cohort of patients and on a subgroup with an intermediate risk of recurrence as defined by the St. Gallen criteria.Conclusion: This study identifies a molecular signature specifying a subgroup of patients who do not gain benefits from tamoxifen treatment. These patients may therefore be eligible for alternative endocrine therapies and/or chemotherapy.

Supplementary Data from CIP2A Is Associated with Human Breast Cancer Aggressivity

Purpose: To investigate the clinical relevance of the recently characterized human oncoprotein cancerous inhibitor of protein phosphatase 2A (CIP2A) in human breast cancer.Experimental Design: CIP2A expression (mRNA and protein) was measured in three different sets of human mammary tumors and compared with clinicopathologic variables. The functional role of CIP2A in breast cancer cells was evaluated by small interfering RNA–mediated depletion of the protein followed by an analysis of cell proliferation, migration, anchorage-independent growth, and xenograft growth.Results: CIP2A mRNA is overexpressed (n = 159) and correlates with higher Scarff-Bloom-Richardson grades (n = 251) in samples from two independent human breast cancer patients. CIP2A protein was found to be overexpressed in 39% of 33 human breast cancer samples. Furthermore, CIP2A mRNA expression positively correlated with lymph node positivity of the patients and with the expression of proliferation markers and p53 mutations in the tumor samples. Moreover, CIP2A protein expression was induced in breast cancer mouse models presenting mammary gland–specific depletion of p53 and either BRCA1 or BRCA2. Functionally, CIP2A depletion was shown to inhibit the expression of its target protein c-Myc. Loss of CIP2A also inhibited anchorage-independent growth in breast cancer cells. Finally, CIP2A was shown to support MDA-MB-231 xenograft growth in nude mice.Conclusions: Our data show that CIP2A is associated with clinical aggressivity in human breast cancer and promotes the malignant growth of breast cancer cells. Thus, these results validate the role of CIP2A as a clinically relevant human oncoprotein and warrant further investigation of CIP2A as a therapeutic target in breast cancer treatment. (Clin Cancer Res 2009;15(16):5092–100)

Supplementary Data from A Gene Expression Signature that Can Predict the Recurrence of Tamoxifen-Treated Primary Breast Cancer

Following the identification of thieno[2,3-d]pyrimidine-based selective and potent inhibitors of MCL-1, we explored the effect of core swapping at different levels of advancement. During hit-to-lead optimization, X-ray-guided S-N replacement in the core provided a new vector, whose exploration led to the opening of the so-called deep-S2 pocket of MCL-1. Unfortunately, the occupation of this region led to a plateau in affinity and had to be abandoned. As the project approached selection of a clinical candidate, a series of core swap analogues were also prepared. The affinity and cellular activity of these compounds showed a significant dependence on the core structure. In certain cases, we also observed an increased and accelerated epimerization of the atropoisomers. The most potent core replacement analogues showed considerable in vivo PD response. One compound was progressed into efficacy studies and inhibited tumor growth.

Myeloid cell leukemia 1 (Mcl-1) has emerged as an attractive target for cancer therapy. It is an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy. Here we report the discovery of our clinical candidate S64315, a selective small molecule inhibitor of Mcl-1. Starting from a fragment derived lead compound, we have conducted structure guided optimization that has led to a significant (3 log) improvement of target affinity as well as cellular potency. The presence of hindered rotation along a biaryl axis has conferred high selectivity to the compounds against other members of the Bcl-2 family. During optimization, we have also established predictive PD markers of Mcl-1 inhibition and achieved both efficient in vitro cell killing and tumor regression in Mcl-1 dependent cancer models. The preclinical candidate has drug-like properties that have enabled its development and entry into clinical trials.

Improving survival outcomes in adult B-cell acute lymphoblastic leukemia (B-ALL) remains a clinical challenge. Relapsed disease has a poor prognosis despite the use of tyrosine kinase inhibitors (TKIs) for Philadelphia chromosome positive (Ph+ ALL) cases and immunotherapeutic approaches, including blinatumomab and chimeric antigen receptor T cells. Targeting aberrant cell survival pathways with selective small molecule BH3-mimetic inhibitors of BCL-2 (venetoclax, S55746), BCL-XL (A1331852), or MCL1 (S63845) is an emerging therapeutic option. We report that combined targeting of BCL-2 and MCL1 is synergistic in B-ALL in vitro. The combination demonstrated greater efficacy than standard chemotherapeutics and TKIs in primary samples from adult B-ALL with Ph+ ALL, Ph-like ALL, and other B-ALL. Moreover, combined BCL-2 or MCL1 inhibition with dasatinib showed potent killing in primary Ph+ B-ALL cases, but the BH3-mimetic combination appeared superior in vitro in a variety of Ph-like ALL samples. In PDX models, combined BCL-2 and MCL1 targeting eradicated ALL from Ph− and Ph+ B-ALL cases, although fatal tumor lysis was observed in some instances of high tumor burden. We conclude that a dual BH3-mimetic approach is highly effective in diverse models of high-risk human B-ALL and warrants assessment in clinical trials that incorporate tumor lysis precautions.

Improving outcomes in acute myeloid leukemia (AML) remains a major clinical challenge. Overexpression of pro-survival BCL-2 family members rendering transformed cells resistant to cytotoxic drugs is a common theme in cancer. Targeting BCL-2 with the BH3-mimetic venetoclax is active in AML when combined with low-dose chemotherapy or hypomethylating agents. We now report the pre-clinical anti-leukemic efficacy of a novel BCL-2 inhibitor S55746, which demonstrates synergistic pro-apoptotic activity in combination with the MCL1 inhibitor S63845. Activity of the combination was caspase and BAX/BAK dependent, superior to combination with standard cytotoxic AML drugs and active against a broad spectrum of poor risk genotypes, including primary samples from patients with chemoresistant AML. Co-targeting BCL-2 and MCL1 was more effective against leukemic, compared to normal hematopoietic progenitors, suggesting a therapeutic window of activity. Finally, S55746 combined with S63845 prolonged survival in xenograft models of AML and suppressed patient-derived leukemia but not normal hematopoietic cells in bone marrow of engrafted mice. In conclusion, a dual BH3-mimetic approach is feasible, highly synergistic, and active in diverse models of human AML. This approach has strong clinical potential to rapidly suppress leukemia, with reduced toxicity to normal hematopoietic precursors compared to chemotherapy.

Background: Recent randomized trials have demonstrated improvements in overall survival (OS) for the BCL-2 inhibitor venetoclax (VEN) in combination with azacitidine and low dose cytarabine in older unfit patients with AML. Pre-clinical studies identified BAX deficiency as a potential mechanism of VEN resistance in AML, but this has not been observed in patients to date (Chen, Cancer Disc 2019). Methods: Patient samples were derived from studies approved by the Alfred Ethics Committee. BAX sequencing was performed using targeted sequencing. In vivo studies used NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. S63845 and S55746 were obtained from Servier/Novartis, A1155463 from G. Lessene (WEHI), venetoclax and cytarabine from Selleckchem. OCI-AML cells were used for protein, viability and competition studies. Results: We used targeted sequencing to assess BAX among 44 patients relapsing after attaining initial remission on VEN-containing regimens for AML. BAX variants at relapse were identified in 6 patients (13.6%), with a variant allelic frequency (VAF) of 0.75% - 48% (Fig A). This included missense, frameshift (fs), nonsense and splice site mutations (Fig A). One variant, P168A, residing in the linker region prior to the α9 helix is known to reduce BAX translocation and activity. The emergence of BAX mutations at AML progression implicated its role in adaptive resistance (Fig B). In contrast, no BAX mutations were identified in a control cohort of 35 patients with AML relapsing after conventional chemotherapy (Fig A). To explore mechanisms of acquired resistance to BH3 mimetics, OCI-AML3 cells were exposed to increasing concentrations of BCL-2 and MCL1 inhibitors over 3-months. Acquired resistance (OCI-AML3-R) was associated with loss of BAX expression, with no changes observed in other BCL-2 family members (Fig C). In contrast, BAX expression was preserved in OCI-AML3 cells exposed to similar concentrations of DMSO (OCI-AML3 DMSO). Targeted BAX NGS revealed a BAX E41Gfs variant in exon 3 at baseline (VAF 46%). In OCI-AML3-R cells, this variant was detected at a VAF of 86%, with no copy number change detected on sequencing-based copy number assessment, consistent with development of loss of heterozygosity in OCI-AML3-R cells. OCI-AML3-R cells were also cross-resistant to an MCL1 inhibitor (S63845), combination of VEN with S63845 or triple combination with VEN, S63845 and a BCL-X inhibitor A1155463 (not shown). In a xenograft model of AML, cohorts transplanted with OCI-AML-R cells lacking wildtype BAX displayed reduced survival compared to mice transplanted with OCI-AML3-DMSO cells after combined treatment with VEN and S63845 (Fig D). As mitochondrial apoptosis is mediated by pro-apoptotic effectors BAX and BAK, we generated OCI-AML3 cells deficient in BAX or BAK by CRISPR/CAS9 gene editing. Only BAX-/- but not BAK-/- cells were resistant to cell death induced by inhibitors of BCL-2 and MCL1 (not shown). This specificity was confirmed in xenograft models of BAX-/- or BAK-/- AML, which confirmed resistance of BAX deficient cells to combined therapy with VEN and S63845 in vivo (Fig E). Finally, to determine if BAX deficiency confers generalized chemoresistance, a competition assay compared proportions of wildtype BAX expressing and BAX-/- OCI-AML3 cells during a 14-day exposure to either 1) VEN, 2) S63845, 3) combined VEN and S63845 or 4) Ara-C. VEN therapy led to expansion of BAX-/- deficient cells, which was enhanced in combination with S63845. In contrast, treatment with Ara-C depleted BAX-/- cells over 4 days, with death of both BAX and BAX-/- populations seen by day 7 (Fig F). Conclusion: We identified the presence of BAX mutations in AML samples from patients progressing on VEN-containing regimens. We show that BAX, but not BAK loss in an AML cell line is associated with resistance to BH3-mimetic drug combinations resulting in reduced survival in AML xenograft models. In contrast, BAX deficiency does not impede the cytotoxic actions of conventional chemotherapy. We conclude that selection of BAX deficient cells may represent a novel mechanism of resistance to BH3-mimetics in the treatment of AML and that the emergence of BAX variants should be considered in patients developing adaptive resistance to VEN-based therapies. Disclosures Moujalled: Walter and Eliza Hall Institute: Patents & Royalties: milestone and royalty payments related to venetoclax.; Servier: Research Funding. Brown:Servier: Research Funding. Anstee:Walter and Eliza Hall Institute: Patents & Royalties: milestone and royalty payments related to venetoclax.. Banquet:Servier: Current Employment. Chanrion:Servier: Current Employment. Maragno:Servier: Current Employment. Schoumacher:Servier: Current Employment. Lessene:Servier: Research Funding; Walter and Eliza Hall Institute: Patents & Royalties: milestone and royalty payments related to venetoclax.. Geneste:Servier: Current Employment. Huang:Genentech: Research Funding; Walter and Eliza Hall Institute: Patents & Royalties: milestone and royalty payments related to venetoclax.; Servier: Research Funding. Roberts:Servier: Research Funding; Janssen: Research Funding; Genentech: Patents & Royalties: for venetoclax to one of my employers (Walter & Eliza Hall Institute); I receive a share of these royalties; AbbVie: Research Funding. Blombery:Janssen: Honoraria; Invivoscribe: Honoraria; Amgen: Consultancy; Novartis: Consultancy. Wei:Genentech: Honoraria, Other: Advisory committee; Servier: Consultancy, Honoraria, Other: Advisory committee; Walter and Eliza Hall Institute: Patents & Royalties; Macrogenics: Honoraria, Other: Advisory committee; AMGEN: Honoraria, Other: Advisory committee, Research Funding; Astra-Zeneca: Honoraria, Other: Advisory committee, Research Funding; Janssen: Honoraria, Other; Abbvie: Honoraria, Other: Advisory committee, Research Funding, Speakers Bureau; Astellas: Honoraria, Other: Advisory committee; Pfizer: Honoraria, Other: Advisory committee; Celgene: Honoraria, Other: Advisory committee, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau.

Avoidance of apoptosis is critical for the development and sustained growth of tumours. The pro-survival protein myeloid cell leukemia 1 (MCL1) is overexpressed in many cancers, but the development of small molecules targeting this protein that are amenable for clinical testing has been challenging. Here we describe S63845, a small molecule that specifically binds with high affinity to the BH3-binding groove of MCL1. Our mechanistic studies demonstrate that S63845 potently kills MCL1-dependent cancer cells, including multiple myeloma, leukaemia and lymphoma cells, by activating the BAX/BAK-dependent mitochondrial apoptotic pathway. In vivo, S63845 shows potent anti-tumour activity with an acceptable safety margin as a single agent in several cancers. Moreover, MCL1 inhibition, either alone or in combination with other anti-cancer drugs, proved effective against several solid cancer-derived cell lines. These results point towards MCL1 as a target for the treatment of a wide range of tumours.

International audience; Escape from apoptosis is one of the major hallmarks of cancer cells. The B-cell Lymphoma 2 (BCL-2) gene family encodes pro-apoptotic and anti-apoptotic proteins that are key regulators of the apoptotic process. Overexpression of the pro-survival member BCL-2 is a well-established mechanism contributing to oncogenesis and chemoresistance in several cancers, including lymphoma and leukemia. Thus, BCL-2 has become an attractive target for therapeutic strategy in cancer, as demonstrated by the recent approval of ABT-199 (Venclexta™) in relapsed or refractory Chronic Lymphocytic Leukemia with 17p deletion. Here, we describe a novel orally bioavailable BCL-2 selective and potent inhibitor called S55746 (also known as BCL201). S55746 occupies the hydrophobic groove of BCL-2. Its selectivity profile demonstrates no significant binding to MCL-1, BFL-1 (BCL2A1/A1) and poor affinity for BCL-XL. Accordingly, S55746 has no cytotoxic activity on BCL-XL-dependent cells, such as platelets. In a panel of hematological cell lines, S55746 induces hallmarks of apoptosis including externalization of phosphatidylserine, caspase-3 activation and PARP cleavage. Ex vivo, S55746 induces apoptosis in the low nanomolar range in primary Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma patient samples. Finally, S55746 administered by oral route daily in mice demonstrated robust anti-tumor efficacy in two hematological xenograft models with no weight lost and no change in behavior. Taken together, these data demonstrate that S55746 is a novel, welltolerated BH3-mimetic targeting selectively and potently the BCL-2 protein.

The development of BH3 mimetics, which antagonize prosurvival proteins of the BCL-2 family, represents a potential breakthrough in cancer therapy. Targeting the prosurvival member MCL-1 has been an area of intense interest because it is frequently deregulated in cancer. In breast cancer, MCL-1 is often amplified, and high expression predicts poor patient outcome. We tested the MCL-1 inhibitor S63845 in breast cancer cell lines and patient-derived xenografts with high expression of MCL-1. S63845 displayed synergistic activity with docetaxel in triple-negative breast cancer and with trastuzumab or lapatinib in HER2-amplified breast cancer. Using S63845-resistant cells combined with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9) technology, we identified deletion of BAK and up-regulation of prosurvival proteins as potential mechanisms that confer resistance to S63845 in breast cancer. Collectively, our findings provide a strong rationale for the clinical evaluation of MCL-1 inhibitors in breast cancer.

Acute myeloid leukemia (AML) is an aggressive and heterogeneous hematologic malignancy, characterized by uncontrolled proliferation and impaired differentiation of myeloid cells. With the exception of certain subtypes, the average long-term survival rate remains low, thus underlining the need to further improve the outcome of AML patients. Since AML is one of the least mutated cancer types, the majority of AML patients may not carry targetable genetic alterations. However, the anti-apoptotic proteins of the Bcl-2 family, such as Bcl-2 and Mcl-1, are often overexpressed in AML, allowing deregulated survival; hence pro-apoptosis priming with small molecule inhibitors of Bcl-2 and Mcl-1 may provide a broader therapeutic benefit across the disease. In addition, a majority of AML patients carry wild-type p53, providing therapeutic opportunity for Hdm2 inhibitors to stabilize p53 and lead to expression of pro-apoptotic molecules (e.g., PUMA & BAX). Therefore, targeting the combined apoptosis mechanisms by inhibiting different anti-apoptotic Bcl-2 family of proteins and activating p53 concomitantly may synergistically enhance apoptotic cell death of AML tumor cells. We tested the combination of Bcl-2 inhibitors (BCL201/S55746 or venetoclax) with either MIK665/S64315, a novel and selective inhibitor of Mcl-1 or HDM201, a selective small molecule inhibitor of p53:Hdm2 interaction, in a series of in vitro and in vivo studies in AML. In vitro, strong combination synergy was observed with a remarkable induction of cell death for both combinations. In vivo, the combination of Bcl-2 inhibitors with MIK665/S64315 or HDM201 lead to complete and durable antitumor responses in a variety of p53wt AML patient-derived xenograft models of heterogeneous genetic profiles. Notably, lowering the dose of HDM201 by 4 fold from its most efficacious dose, resulted in a high degree of tumor regressions while mitigating the toxicity effects on platelets. Taken together, these data demonstrate that a combination of Bcl-2 inhibitor (BCL201/S55746 or venetoclax) with MIK665/S64315 or HDM201 provide therapeutic benefit over the monotherapy, and support a rationale for testing these apoptosis enhancing combination approaches in AML patients. Citation Format: Youzhen Wang, Shumei Qui, Sneha Sanghavi, Iain Mulford, Gaëlle Lysiak, Maïa Chanrion, Prakash Mistry, Ulrike Pfaar, Marie Schoumacher, Audrey Claperon, Laurence Kraus-Berthier, Sébastien Banquet, Alix Derreal, Claire Fabre, Heiko Maacke, Frédéric Colland, Olivier Geneste, Erick Morris, Ensar Halilovic. Targeting AML through apoptosis activation using Bcl-2/Mcl-1 or Bcl-2/Hdm2 inhibitor combination therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 257.

Mcl-1 is highly expressed in a variety of human cancers (including those of hematopoietic and lymphoid origin) and is exploited by cancer cells to evade cell death and to develop resistance to diverse chemotherapeutic agents. We disclose, for the first time, the structure of S64315 (also named MIK665) a highly potent and selective inhibitor of Mcl-1 with improved potency over its predecessor S63845 (Kotschy et al, Nature, 2016). S64315/MIK665 is currently in phase 1 in AML (Acute Myeloid Leukemia) and MDS (Myelodysplastic Syndrome) (EudraCT 2016-003768-38, NCT 02979366) and in MM (Multiple Myeloma) and lymphoma (NCT02992483). A fragment-based, structure-guided drug discovery effort led to the identification of S64315/MIK665 that binds to human Mcl-1 with a sub-nanomolar affinity (Ki 0.048 nM) and selectively over other anti-apoptotic Bcl-2 family members. It has similar affinity for human, rat, dog and monkey Mcl-1 but about a ten-fold lower affinity for mouse Mcl-1. S64315/MIK665 causes dose-dependent activation of the intrinsic apoptosis pathway in a Bax/Bak-dependent manner, as measured by increased caspase activity and cleaved PARP. S64315/MIK665 shows strong cell killing activity in a diverse panel of human hematological tumor cell lines, including AML, lymphoma and MM. The activity profile of S64315/MIK665 is distinct from that of venetoclax, a selective Bcl2 inhibitor. In vivo, S64315 as single agent demonstrated potent and dose-dependent apoptotic and antitumor response after intravenous administration in several human hematological tumor models grafted in immuno-compromised mice and rats. Complete regression of established tumors, at well tolerated doses, was achieved using different intravenous dosing regimens in rats as well as in mice. Finally, dual BH3-mimetic targeting approach combining S64315/MIK665 with BCL2 inhibitors showed strong and durable antitumor responses in several hematological tumor models both in vitro and in vivo. Citation Format: Ana Leticia Maragno, Prakash Mistry, András Kotschy, Zoltán Szlavik, James Murray, James Davidson, Gaëtane Le Toumelin-Braizat, Maïa Chanrion, Alain Bruno, Audrey Claperon, Heiko Maacke, Erick Morris, Youzhen Wang, Alix Derreal, Márton Csekei, Attila Paczal, Zoltán Szabo, Szabolcs Sipos, Agnes Proszenyak, Balázs Balint, Allan Surgenor, Pawel Dokurno, Natalia Matassova, Ijen Chen, Gaëlle Lysiak-Auvity, Anne-Marie Girard, Fabienne Grave, Frédéric Colland, Ensar Halilovic, Olivier Geneste. S64315 (MIK665) is a potent and selective Mcl1 inhibitor with strong antitumor activity across a diverse range of hematologic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4482.

One of the hallmarks of cancer is evasion of apoptosis. The B-cell lymphoma-2 (Bcl-2) family of proteins represents a crucial point of control of apoptosis. The Bcl-2 family comprises both pro- and anti-apoptotic members, the latter of which (Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and Bcl-2A1) are often overexpressed in cancer cells, supporting their aberrant survival. Thus, these anti-apoptotic proteins have become an attractive target for cancer therapy. BH3 mimetics have been shown to bind to the BH3 binding groove of anti-apoptotic Bcl-2 family members and inhibit their function, resulting in apoptotic cell death, and one such BH3 mimetic, ABT-199 (venetoclax), has recently been approved for treatment of relapsed or refractory Chronic Lymphocytic Leukemia. We have developed two novel and potent BH3 mimetics: MIK665/S64315, a highly selective inhibitor of Mcl-1 and BCL201/S55746, a selective Bcl-2 inhibitor. Both compounds, individually induce apoptosis in hematological cancer cell lines, primary patient samples and demonstrate anti-tumor efficacy in xenograft models. MIK665/S64315 is currently in phase 1 clinical development in AML and MDS (NCT 02979366) and in MM and lymphoma (NCT02992483). Here, we describe the activity of the combination of MIK665/S64315 with BCL201/S55746 or venetoclax, both in vitro and in vivo, across a range of hematological indications (AML, MM and DLBCL). In vitro, a strong synergy was observed with these combinations, resulting in a remarkable induction of cell death in majority of cell lines tested. In vivo, MIK665/S64315 and BCL201/S55746 combinations lead to complete and durable antitumor responses in many different xenograft models in mice and rats. Taken together, these data demonstrate that a combination of MIK665/S64315 and BCL201/S55746 provide strong therapeutic benefit over either monotherapy, and support a rationale for testing Mcl-1 and Bcl-2 inhibitor combinations in patients with hematological malignancies. Citation Format: Ensar Halilovic, Maïa Chanrion, Prakash Mistry, Markus Wartmann, Shumei Qiu, Sneha Sanghavi, Yan Chen, Gaëlle Lysiak, Ana Leticia Maragno, Ulrike Pfaar, Felix Huth, Marie Schoumacher, Audrey Claperon, Laurence Kraus-Berthier, Sébastien Banquet, Alix Derreal, Heiko Maacke, Frédéric Colland, Olivier Geneste, Erick Morris, Youzhen Wang. MIK665/S64315, a novel Mcl-1 inhibitor, in combination with Bcl-2 inhibitors exhibits strong synergistic antitumor activity in a range of hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4477.

Purpose: Receptor-interacting protein of 140 kDa (RIP140) is a transcriptional cofactor for nuclear receptors involved in reproduction and energy homeostasis. Our aim was to investigate its role in the regulation of E2F1 activity and target genes both in breast cancer cell lines and in tumor biopsies. Experimental Design: Glutathione S-transferase pull-down assays, coimmunoprecipitation experiments, and chromatin immunoprecipitation analysis were used to evidence interaction between RIP140 and E2F1. The effects of RIP140 expression on E2F1 activity were determined using transient transfection and quantification of E2F target mRNAs by quantitative real-time PCR. The effect on cell cycle was assessed by fluorescence-activated cell sorting analysis on cells overexpressing green fluorescent protein–tagged RIP140. A tumor microarray data set was used to investigate the expression of RIP140 and E2F1 target genes in 170 breast cancer patients. Results: We first evidenced the complex interaction between RIP140 and E2F1 and showed that RIP140 represses E2F1 transactivation on various transiently transfected E2F target promoters and inhibits the expression of several E2F1 target genes (such as CCNE1 and CCNB2). In agreement with a role for RIP140 in the control of E2F activity, we show that increasing RIP140 levels results in a reduction in the proportion of cells in S phase in various human cell lines. Finally, analysis of human breast cancers shows that low RIP140 mRNA expression was associated with high E2F1 target gene levels and basal-like tumors. Conclusion: This study shows that RIP140 is a regulator of the E2F pathway, which discriminates luminal- and basal-like tumors, emphasizing the importance of these regulations for a clinical cancer phenotype. Clin Cancer Res; 16(11); 2959–70. ©2010 AACR.

Purpose: To investigate the clinical relevance of the recently characterized human oncoprotein cancerous inhibitor of protein phosphatase 2A (CIP2A) in human breast cancer. Experimental Design: CIP2A expression (mRNA and protein) was measured in three different sets of human mammary tumors and compared with clinicopathologic variables. The functional role of CIP2A in breast cancer cells was evaluated by small interfering RNA–mediated depletion of the protein followed by an analysis of cell proliferation, migration, anchorage-independent growth, and xenograft growth. Results: CIP2A mRNA is overexpressed (n = 159) and correlates with higher Scarff-Bloom-Richardson grades (n = 251) in samples from two independent human breast cancer patients. CIP2A protein was found to be overexpressed in 39% of 33 human breast cancer samples. Furthermore, CIP2A mRNA expression positively correlated with lymph node positivity of the patients and with the expression of proliferation markers and p53 mutations in the tumor samples. Moreover, CIP2A protein expression was induced in breast cancer mouse models presenting mammary gland–specific depletion of p53 and either BRCA1 or BRCA2. Functionally, CIP2A depletion was shown to inhibit the expression of its target protein c-Myc. Loss of CIP2A also inhibited anchorage-independent growth in breast cancer cells. Finally, CIP2A was shown to support MDA-MB-231 xenograft growth in nude mice. Conclusions: Our data show that CIP2A is associated with clinical aggressivity in human breast cancer and promotes the malignant growth of breast cancer cells. Thus, these results validate the role of CIP2A as a clinically relevant human oncoprotein and warrant further investigation of CIP2A as a therapeutic target in breast cancer treatment. (Clin Cancer Res 2009;15(16):5092–100)

Many reports support that xenografts from patient-derived xenografts (PDXs) in mice or rats well recapitulate the molecular diversity, cellular heterogeneity, and histology seen in patient tumors. However, shortcomings including limited clinical diversity of the PDXs, absence of influence of human microbiome, absence of drug human metabolism and reduced immune system restrain the predictive values of these PDX models. To set-up a holistic integration of these criticisms, we have associated efforts from public hospitals, academic groups, biotechs and private pharmaceutical companies with the financial support of the French Ministry of Industry. Patient surgical specimens from different breast tumor subtypes, among 9 other cancer pathologies, are collected to establish large collections of PDXs in mice. In addition, in vitro primary cultures of cells from these samples are conducted to establish a collection of cell lines from the stromal and the tumoral compartments. The 40 established breast PDX models represent the 4 major molecular clinical subtypes among which 37% are triple negative, 33% are Luminal B HER2-, 22% are Luminal B Her2+ and 8% Her2+. The models are evaluated for ex vivo and in vivo sensitivities to 4 clinically relevant anticancer drugs, for histology, immune infiltrates, and molecular characteristics including the analysis of gene polymorphism, RNAseq gene expression and NGS exome sequencing of 112 genes. The metagenomic sequencing of the gut microbiota from stools collected in both patients and tumor-bearing mice before and after chemotherapeutic treatments improves our knowledge on the role of microbiota in cancer progression and treatment. Secondly, to increase the predictability of the models, we are generating mice with humanized liver showing distinct drug pharmacokinetic profiles as compared with parental mice. Finally, the humanization of the immune system in mice is developed by several approaches including the use of induced pluripotent stem cells from cancer patients. These tumor collection and model characterization were performed under harmonized procedures within the consortium, allowing high quality material and results. All model characteristics are being compiled in a web-based database for efficient features search and data mining. We will present the first characterized breast cancer models and will discuss their usefulness and chance to bring benefit to patients via this holistic strategy developed within the IMODI initiative. Citation Format: Olivier Duchamp, Severine Tabone-Eglinger, Caroline Mignard, Isabelle Goddard, Alain Bruno, Maia Chanrion, Loreley Calvet, Olivier Degoul, Kevin Dhondt, Liliane Goetsch, Julie Miralves, Christophe Lautrette, Cindy Pensec, Françoise Le Vacon, Isabelle Treilleux, Laurent Arnould, Philippe Vaglio, Grégoire Prevost, Olivier Tredan. Innovative and predictive models against breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5015. doi:10.1158/1538-7445.AM2017-5015

Purpose: The identification of a molecular signature predicting the relapse of tamoxifen-treated primary breast cancers should help the therapeutic management of estrogen receptor–positive cancers. Experimental Design: A series of 132 primary tumors from patients who received adjuvant tamoxifen were analyzed for expression profiles at the whole-genome level by 70-mer oligonucleotide microarrays. A supervised analysis was done to identify an expression signature. Results: We defined a 36-gene signature that correctly classified 78% of patients with relapse and 80% of relapse-free patients (79% accuracy). Using 23 independent tumors, we confirmed the accuracy of the signature (78%) whose relevance was further shown by using published microarray data from 60 tamoxifen-treated patients (63% accuracy). Univariate analysis using the validation set of 83 tumors showed that the 36-gene classifier is more efficient in predicting disease-free survival than the traditional histopathologic prognostic factors and is as effective as the Nottingham Prognostic Index or the “Adjuvant!” software. Multivariate analysis showed that the molecular signature is the only independent prognostic factor. A comparison with several already published signatures demonstrated that the 36-gene signature is among the best to classify tumors from both training and validation sets. Kaplan-Meier analyses emphasized its prognostic power both on the whole cohort of patients and on a subgroup with an intermediate risk of recurrence as defined by the St. Gallen criteria. Conclusion: This study identifies a molecular signature specifying a subgroup of patients who do not gain benefits from tamoxifen treatment. These patients may therefore be eligible for alternative endocrine therapies and/or chemotherapy.

The cDNA microarray technology allows the simultaneous analysis of all genes expressed in a tumor. This approach has already permitted to propose a molecular classification of breast cancers. Gene expression profiling is now expected to define prognosis signatures claiming the disease outcome as well as signatures predicting response to therapy. The over-expression of a cluster of genes that are implicated in cell cycle and mitosis control has been shown to discriminate a subgroup of ER+ breast cancers exhibiting a poor prognosis. This "proliferation cluster" is shown to be also present in signatures predicting relapse of ER+ breast cancers treated by the anti-estrogen tamoxifen, including the 36-gene classifier that we have recently identified. Consequently, it seems legitimate to wonder if this part of such predictive signature is really specific to the insensitivity to tamoxifen or rather indicates a poor disease outcome whatever the therapy applied. On the other hand, low expression of ER+ related genes and high expression of genes associated to ER- status or low ERalpha levels have been shown to sign a poor prognosis. Whether the estrogen-related genes that are present in our 36-gene classifier specify the clinical disease outcome or are really specific to the response to tamoxifen, remains to be determined. In any case, the specificity of our 36-gene classifier as those predicting the recurrence of ER+ breast cancer under one treatment or another should be demonstrated. In the same way, future studies should define molecular signatures that will be really predictive of the response to the treatment in order to define which one is the most suitable to decrease the risk of relapse of ER+ breast cancers. These studies should be based on neoadjuvant clinical trials that permit to evaluate the response to treatment in an objective manner.

BackgroundCurrent histo-pathological prognostic factors are not very helpful in predicting the clinical outcome of breast cancer due to the disease's heterogeneity. Molecular profiling using a large panel of genes could help to classify breast tumours and to define signatures which are predictive of their clinical behaviour.MethodsTo this aim, quantitative RT-PCR amplification was used to study the RNA expression levels of 47 genes in 199 primary breast tumours and 6 normal breast tissues. Genes were selected on the basis of their potential implication in hormonal sensitivity of breast tumours. Normalized RT-PCR data were analysed in an unsupervised manner by pairwise hierarchical clustering, and the statistical relevance of the defined subclasses was assessed by Chi2 analysis. The robustness of the selected subgroups was evaluated by classifying an external and independent set of tumours using these Chi2-defined molecular signatures.ResultsHierarchical clustering of gene expression data allowed us to define a series of tumour subgroups that were either reminiscent of previously reported classifications, or represented putative new subtypes. The Chi2 analysis of these subgroups allowed us to define specific molecular signatures for some of them whose reliability was further demonstrated by using the validation data set. A new breast cancer subclass, called subgroup 7, that we defined in that way, was particularly interesting as it gathered tumours with specific bioclinical features including a low rate of recurrence during a 5 year follow-up.ConclusionThe analysis of the expression of 47 genes in 199 primary breast tumours allowed classifying them into a series of molecular subgroups. The subgroup 7, which has been highlighted by our study, was remarkable as it gathered tumours with specific bioclinical features including a low rate of recurrence. Although this finding should be confirmed by using a larger tumour cohort, it suggests that gene expression profiling using a minimal set of genes may allow the discovery of new subclasses of breast cancer that are characterized by specific molecular signatures and exhibit specific bioclinical features.