Preclinical Models Of Triple-negative Breast Cancer Research Articles

A BPTF Inhibitor That Interferes with the Multidrug Resistance Pump to Sensitize Murine Triple-Negative Breast Cancer Cells to Chemotherapy.

Triple-negative breast cancer (TNBC) is associated with a generally poor prognosis due to its highly aggressive and metastatic nature, lack of targetable receptors, as well as the frequent development of resistance to chemotherapy. We previously reported that AU1, a small molecule developed as an inhibitor of BPTF (bromodomain PHD finger-containing transcription factor), was capable of sensitizing preclinical models of TNBC to chemotherapy in part via the promotion of autophagy. In studies reported here, we identify an additional property of this compound, specifically that sensitization is associated with the inhibition of the P-glycoprotein (P-gp) efflux pump. In silico molecular docking studies indicate that AU1 binds to active regions of the efflux pump in a manner consistent with the inhibition of the pump function. This work identifies a novel chemical structure that can influence multidrug efflux, an established mechanism of drug resistance in TNBC, that has not yet been successfully addressed by clinical efforts.

Protease activated receptor-1 regulates mixed lineage kinase-3 to drive triple-negative breast cancer tumorigenesis

Triple-negative breast cancer (TNBC) is difficult to treat breast cancer subtype due to lack or insignificant expressions of targetable estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2). Therefore, finding a targetable protein or signaling pathway in TNBC would impact patient care. Here, we report that a member of the Mixed Lineage Kinase (MLK) family, MLK3, is an effector of G-protein-coupled protease-activated receptors 1 (PAR1) and targeting MLK3 by a small-molecule inhibitor prevented PAR1-mediated TNBC tumorigenesis. In silico and immunohistochemistry analysis of human breast tumors showed overexpression of PAR1 and MLK3 in TNBC tumors. Treating α-thrombin and PAR1 agonist increased MLK3 and JNK activities and induced cell migration in TNBC cells. The PAR1 positive/high (PAR1+/hi) population of TNBC cells showed aggressive tumor phenotype with increased MLK3 signaling. Moreover, combined inhibition of the PAR1 and MLK3 mitigated the TNBC tumor burden in preclinical TNBC models. Our data suggests that activation of the PAR1-MLK3 axis promotes TNBC tumorigenesis. Therefore, combinatorial therapy targeting MLK3 and PAR1 could effectively reduce TNBC tumor burden.

Novel Inhibition of Central Carbon Metabolism Pathways by Rac and CDC42 inhibitor MBQ167 and Paclitaxel.

Triple negative breast cancer (TNBC) represents a therapeutic challenge in which standard chemotherapy is limited to paclitaxel. MBQ167, a clinical stage small molecule inhibitor that targets Rac and Cdc42, inhibits tumor growth and metastasis in mouse models of TNBC. Herein, we investigated the efficacy of MBQ167 in combination with paclitaxel in TNBC preclinical models, as a prelude to safety trials of this combination in patients with advanced breast cancer. Individual MBQ167 or combination therapy with paclitaxel was more effective at reducing TNBC cell viability and increasing apoptosis compared with paclitaxel alone. In orthotopic mouse models of human TNBC (MDA-MB231 and MDA-MB468), individual MBQ167, paclitaxel, or the combination reduced mammary tumor growth with similar efficacy, with no apparent liver toxicity. However, paclitaxel single agent treatment significantly increased lung metastasis, whereas MBQ167, single or combined, reduced lung metastasis. In the syngeneic 4T1/BALB/c model, combined MBQ167 and paclitaxel decreased established lung metastases by ∼80%. To determine the molecular basis for the improved efficacy of the combined treatment on metastasis, 4T1 tumor extracts from BALB/c mice treated with MBQ167, paclitaxel, or the combination were subjected to transcriptomic analysis. Gene set enrichment identified specific downregulation of central carbon metabolic pathways by the combination of MBQ167 and paclitaxel but not individual compounds. Biochemical validation, by immunoblotting and metabolic Seahorse analysis, shows that combined MBQ167 and paclitaxel reduces glycolysis. This study provides a strong rationale for the clinical testing of MBQ167 in combination with paclitaxel as a potential therapeutic for TNBC and identifies a unique mechanism of action.

The therapeutic role of γδT cells in TNBC.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that presents significant therapeutic challenges due to the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. As a result, conventional hormonal and targeted therapies are largely ineffective, underscoring the urgent need for novel treatment strategies. γδT cells, known for their robust anti-tumor properties, show considerable potential in TNBC treatment as they can identify and eliminate tumor cells without reliance on MHC restrictions. These cells demonstrate extensive proliferation both in vitro and in vivo, and can directly target tumors through cytotoxic effects or indirectly by promoting other immune responses. Studies suggest that expansion and adoptive transfer strategies targeting Vδ2 and Vδ1 γδT cell subtypes have shown promise in preclinical TNBC models. This review compiles and discusses the existing literature on the primary subgroups of γδT cells, their roles in cancer therapy, their contributions to tumor cell cytotoxicity and immune modulation, and proposes potential strategies for future γδT cell-based immunotherapies in TNBC.

Entolimod, a clinical stage flagellin derivative, improves clinically relevant chemo-immunotherapy responses in pre-clinical triple negative breast cancer

Abstract Chemotherapy+anti-PD-1 is FDA approved and has improved outcomes for locally advanced and metastatic PD-L1+ triple negative breast cancer (TNBC). Unfortunately, a need still exists to improve this chemo-immunotherapy (CTx-I) platform especially in advanced disease. Entolimod, an agonist of the TLR5 and NAIP5 receptors, is our flagellin derivative which has successfully completed Phase I safety trials. We previously showed that systemically administered entolimod enhances durable antitumor immunity against metastasis of 4T1, a preclinical PD-L1+ TNBC model. However, the antimetastatic activity of single-agent entolimod was not completely effective, highlighting the need to combine entolimod with additional immune-based modalities such as CTx-I to further improve tumor control. Here, we used Paclitaxel+anti-PD-1 as a clinically oriented CTx-I platform in pre-clinical metastatic TNBC models. CTx-I+entolimod effectively controlled primary tumors and reduced lung metastatic burden more so than the single or double agent treatments. Consistent with our prior work that neutrophils express functional TLR5, depletion abrogated CTx-I+entolimod efficacy. Collectively, the immune enhancing effects of entolimod can be leveraged to boost urgently needed CTx-I outcomes in TNBC. Because entolimod has successfully completed Phase I clinical trials, this preclinical work supports the rationale design of a clinical trial that combines CTx-I with entolimod as an effective immunotherapy platform.

Pharmacological suppression of the OTUD4/CD73 proteolytic axis revives antitumor immunity against immune-suppressive breast cancers.

Despite widespread utilization of immunotherapy, treating immune-cold tumors remains a challenge. Multiomic analyses and experimental validation identified the OTUD4/CD73 proteolytic axis as a promising target in treating immune-suppressive triple negative breast cancer (TNBC). Mechanistically, deubiquitylation of CD73 by OTUD4 counteracted its ubiquitylation by TRIM21, resulting in CD73 stabilization inhibiting tumor immune responses. We further demonstrated the importance of TGF-β signaling for orchestrating the OTUD4/CD73 proteolytic axis within tumor cells. Spatial transcriptomics profiling discovered spatially resolved features of interacting malignant and immune cells pertaining to expression levels of OTUD4 and CD73. In addition, ST80, a newly developed inhibitor, specifically disrupted proteolytic interaction between CD73 and OTUD4, leading to reinvigoration of cytotoxic CD8+ T cell activities. In preclinical models of TNBC, ST80 treatment sensitized refractory tumors to anti-PD-L1 therapy. Collectively, our findings uncover what we believe to be a novel strategy for targeting the immunosuppressive OTUD4/CD73 proteolytic axis in treating immune-suppressive breast cancers with the inhibitor ST80.

Abstract 7592: Combined inhibition of CDK4/6 and AKT is highly active against the luminal androgen receptor (LAR) subtype of triple negative breast cancer (TNBC)

Abstract The LAR subtype of TNBC is enriched for targetable biomarkers such as androgen receptor (AR) expression, PIK3CA mutations, and intact Rb. The purpose of this study was to investigate the most effective combination of CDK4/6, AR, and PI3K-AKT inhibitors in preclinical models of LAR TNBC. The MDAMB453 and MFM223 (AR positive/Rb intact/PIK3CAmutant) LAR TNBC cells were treated with each inhibitor alone or in combination. Drug sensitivity was assessed using colony formation, cell viability, and live-cell analysis. The Loewe additivity equation was used to measure the additive drug effects. Immunoblot analysis was used to evaluate cell cycle and PI3K-AKT signaling and ARE luciferase assay was used to assess AR transcriptional activity. MDAMB453 and MFM223 cells were sensitive to the single agents palbociclib, alpelisib, and capivasertib (IC50, ~500 nM), which inhibited CDK4/6, PI3Kα, and AKT, respectively. Enzalutamide, an AR antagonist, exerted no growth inhibitory activity (IC50, 15-25 μM). Treatment with palbociclib, alpelisib, or capivasertib did not alter the AR protein levels or transcriptional activity. The combination of palbociclib+capivasertib synergistically inhibited the proliferation of MDAMB453 and MFM223 cells (synergistic scores 7.34, p=5.81-11, and 4.78, p=0.012, respectively) better than palbociclib+alpelisib. Enzalutamide did not enhance either combination. Palbociclib+capivasertib inhibited PI3KCA-mutant and Rb-intact LAR TNBC PDX growth more potently than palbociclib+alpelisib (p=0.046). Treatment of LAR TNBC cells with palbociclib suppressed Rb phosphorylation and resulted in adaptive activation of P-AKTS473/T308 and the AKT substrates GSK3β, PRAS40, and FoxO3a after 24 h. Capivasertib inhibited the phosphorylation of AKT and AKT substrates, following CDK4/6 blockade with palbociclib, more potently than alpelisib. PI3Kβ inhibitors (TGX221, GSK2636771) did not block AKT substrate upregulation, suggesting PI3Kβ does not mediate adaptive responses. Rictor knockdown failed to reduce AKT activation following palbociclib treatment, suggesting an mTORC2-independent mechanism. Human Phospho-kinase Array of MDA-MB-453 cells treated with palbociclib showed time dependent upregulation of GSK3βS9, STAT3Y727 and PDFGRβY751. PDGFRβ activation after palbociclib treatment was confirmed in MFM223 cells. PDGFRβ siRNAs in MDAMB453 cells blocked P-AKTS473 upregulation after palbociclib treatment. Treatment with palbociclib and the PDGFRβ small-molecule antagonist CP673451 arrested LAR TNBC growth similar to palbociclib+capivasertib, suggesting an adaptive response involving PDGFRβ signaling. In conclusion, this study supports the clinical testing of the palbociclib and capivasertib combination for LAR TNBC patients and reveals adaptive responses after palbociclib treatment through PDGFRβ signaling. Citation Format: Rosario Chica-Parrado, Gun Min Kim, Yasuaki Uemoto, Dan Ye, Fabiana Napolitano, Chang-Ching Lin, Emmanuel Bikorimana, Kyung-min Lee, Saurabh Mendiratta, Ariella B. Hanker, Carlos L. Arteaga. Combined inhibition of CDK4/6 and AKT is highly active against the luminal androgen receptor (LAR) subtype of triple negative breast cancer (TNBC) [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 7592.

Targeting Receptor Tyrosine Kinases as a Novel Strategy for the Treatment of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) comprises a group of aggressive and heterogeneous breast carcinoma. Chemotherapy is the mainstay for the treatment of triple-negative tumors. Nevertheless, the success of chemotherapeutic treatments is limited by their toxicity and development of acquired resistance leading to therapeutic failure and tumor relapse. Hence, there is an urgent need to explore novel targeted therapies for TNBC. Receptor tyrosine kinases (RTKs) are a family of transmembrane receptors that are key regulators of intracellular signaling pathways controlling cell proliferation, differentiation, survival, and motility. Aberrant activity and/or expression of several types of RTKs have been strongly connected to tumorigenesis. RTKs are frequently overexpressed and/or deregulated in triple-negative breast tumors and are further associated with tumor progression and reduced survival in patients. Therefore, targeting RTKs could be an appealing therapeutic strategy for the treatment of TNBC. This review summarizes the current evidence regarding the antitumor activity of RTK inhibitors in preclinical models of TNBC. The review also provides insights into the clinical trials evaluating the use of RTK inhibitors for the treatment of patients with TNBC.

Abstract LB_C06: Utilizing a novel HDAC inhibitor bocodepsin (OKI-179) to overcome doxorubicin resistance in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic treatment options and a high rate of metastatic recurrence. TNBC is defined by lack of estrogen and progesterone receptor expression with non-amplified HER2, and accounts for 10-15% of all breast cancer cases. While pembrolizumab, sacituzumab govitecan and PARP inhibitors are beneficial for a subset of TNBC patients, chemotherapy including doxorubicin continues to be the primary treatment. Bocodepsin is a novel, orally bioavailable clinical stage Class 1-targeting HDAC inhibitor with activity in preclinical models of TNBC. The purpose of this study was to investigate OKI-005 (in vitro) and bocodepsin (in vivo) in combination with doxorubicin (DOX) to overcome resistance. Procedures: TNBC cell lines were exposed to a no drug, DOX, OKI-005 or combination for 72 hours and cell viability was assessed via CellTiter-Glo. Synergy scores were calculated from three independent replicates using Synergy Finder+. Apoptosis was assessed following 48 hours of drug exposure by flow cytometry. Immunoblots were performed following 24 hours of drug treatment to evaluate mechanism of action. In vitro senescence-associated beta-galactosidase was performed after 6 days drug treatment. For in vivo studies, 5 million MDA-MB-231 cells were injected subcutaneously in athymic nude mice. Mice were treated with either no drug, 1.5 mg/kg DOX IP QW, 80 mg/kg bocodepsin PO QD, or the combination. Results: Bliss scores calculated from Synergy Finder+ using the CellTiter-Glo data showed synergy for the combination of OKI-005 and DOX in all TNBC cell lines. The combination resulted in statistically significant increased apoptosis measured by Annexin V compared to single agent drug treatment in CAL-51, MDA-MB-231 and CAL-120 cell lines. Immunoblot analysis demonstrated an increase in the pro-apoptotic protein BIM and decrease in anti-apoptotic protein BCL-XL with the combination. The cyclin dependent kinase inhibitor p21 was increased by OKI-005 independently of p53 and doxorubicin for the cell lines MDA-MB-231, Hs 578T, and CAL-120. The combination resulted in increased cleaved caspase-3 for all cell lines evaluated. Senescence-associated beta-galactosidase showed an increase in senescent cells for both DOX and OKI-005, which was eradicated in combination. DOX and bocodepsin were further tested in vivo, where significant tumor growth inhibition was observed in combination compared to DOX (p=0.0016) and bocodepsin (p=0.0068). Conclusion: The addition of OKI-005 or bocodepsin to doxorubicin resulted in improved anti-proliferative and anti-cancer activity with evidence of increased apoptosis and decreased senescence. As a result, bocodepsin is a promising combination partner for doxorubicin to overcome doxorubicin resistance and promote apoptosis in TNBC. Bocodepsin has a favorable toxicity profile compared to other HDAC inhibitors and this work supports the potential clinical investigation of doxorubicin and bocodepsin in patients with metastatic TNBC. Citation Format: Stephen G Smoots, Anna R Schreiber, Marilyn M Jackson, Evan D Dus, Stacey M Bagby, Adrian T A. Dominguez, Cameron A Binns, Jennifer R Diamond, Todd M Pitts. Utilizing a novel HDAC inhibitor bocodepsin (OKI-179) to overcome doxorubicin resistance in triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C06.

Abstract LB_C07: Utilizing a BCL-2 inhibitor (venetoclax) to overcome doxorubicin resistance in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of the progesterone receptor, estrogen receptor, and human epidermal growth factor-2 over-expression. TNBC comprises 10-15% of all breast cancer cases and compared to other subtypes of breast cancer, TNBC is associated with a higher risk for metastatic recurrence and the chemotherapy agent doxorubicin (dox) remains a mainstay of treatment. Senescence is a cellular phenomenon where cells are committed to an arrested state, however, senescent cells can secrete pro-tumorigenic factors which can help to promote tumor progression and invasion. Senescence is one potential mechanism of resistance to doxorubicin in breast cancer. Avoiding senescence-mediated resistance and promoting apoptosis is one strategy to improve response to dox in TNBC. Venetoclax is an orally bioavailable FDA approved BCL-2 inhibitor with activity in preclinical models of TNBC. The purpose of this study was to investigate venetoclax in combination with dox to overcome senescence-mediated resistance and induce apoptosis. Experimental Procedures: TNBC cell lines were exposed to a no drug, dox, venetoclax or combination at increasing doses for 72 hours and cell viability was assessed via CellTiter-Glo. Synergy scores were calculated from three independent replicates using Synergy Finder+. Apoptosis at 48 hours was analyzed by flow cytometry using Annexin V on cells treated in combination and with single agents. In vitro cell survival following drug treatment for 24 hours was assessed via clonogenic assay. In vitro senescence-associated β-gal was performed after 6 days drug treatment. Athymic nude mice were subcutaneously injected with 5 million MDA-MB-231 cells for in vivo studies. Mice were treated with either no drug, 60 mg/kg venetoclax PO QD, 1.5 mg/kg dox IP QW, or the combination. Results: The combination of venetoclax and dox resulted in synergy based on CellTiter-Glo data, which was used to calculate Bliss scores using Synergy Finder+ in CAL-51, MDA-MB-231, and CAL-120 TNBC cell lines. The dox and venetoclax combination caused a statistically significant increase in apoptosis measured by Annexin V staining when compared to single agents and control in all cell lines. The combination resulted in a decrease in cellular survival in all cell lines tested as measured by the clonogenic assay. Senescence-associated beta-galactosidase showed an increase in senescent cells for both dox and venetoclax treated cells, which was eradicated in combination. Dox and venetoclax were further tested in vivo, where significant tumor growth inhibition was observed in combination compared to doxorubicin (p = 0.0031) and venetoclax alone (p = 0.0038) after 48 days of treatment. Conclusion: The addition of venetoclax to doxorubicin resulted in decreased cellular proliferation, increased apoptosis, and decreased senescence. As a result, venetoclax is a promising combination partner for doxorubicin to overcome doxorubicin resistance and promote apoptosis in TNBC. Citation Format: Evan D Dus, Stephen G Smoots, Anna R Schreiber, Marilyn M Jackson, Stacey M Bagby, Adrian T A Dominguez, Cameron A Binns, Todd M Pitts, Jennifer R Diamond. Utilizing a BCL-2 inhibitor (venetoclax) to overcome doxorubicin resistance in triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C07.

Abstract LB_C04: Novel orally bioavailable macrocycles that target cyclin A and B elicit antitumor activity in breast cancer patient-derived xenograft models

Abstract Background: Cyclin/cyclin-dependent kinase (CDK) complexes regulate the activity of RB1 and E2F to drive cell cycle progression while ensuring fidelity of DNA replication. The interaction between cyclins and a critical subset of their substrates, including RB1 and E2F, is mediated by a conserved short linear RxL motif. RxL peptides have been shown to inhibit substrate phosphorylation by CDK2/cyclin A (AACR 2022 poster #5379). Previously, we have shown that macrocycles that selectively inhibit RxL-mediated binding to cyclins A and B (RxL inhibitors) lead to growth inhibitory activity in multiple cancer cell lines, and result in tumor regression in small cell lung cancer (SCLC) and ovarian cancer xenograft models (AACR 2023 poster #1560). Here, we test the antitumor activity of CID-016 in patient-derived xenograft (PDX) models from breast cancer. Methods: Bioinformatic analyses using RNA sequencing (RNAseq) and whole exome sequencing (WES) data from 39 breast cancer cell lines was employed to identify biomarker profiles that associate with sensitivity to CID-016. The antiproliferative activity of CID-016 was assessed by GI50 after 4-8 days of treatment. Hallmark pathway scores were calculated by Gene Set Variation Analysis (GSVA) method using MSigDb Hallmark collection from cell lines and existing PDX models. Based on the calculated hallmark pathway score, triple negative breast cancer (TNBC) PDX models were selected to test the anti-tumor activity of CID-016. In vivo, NMRI-Foxn1nu/nu mice were implanted with tumor fragments in the lower flank and treated with vehicle or CID-016 oral formulation of 50-100 mg/kg BID or 100 mg/kg QD, respectively for at least 28 days. Tumor volume and body weight were measured biweekly. Pharmacodynamic (PD) analyses will be conducted after treatment with CID-016. Results: Treatment of the breast cancer cell line panel with CID-016 demonstrated greater potency by GI50 in TNBC compared to other breast cancer cell lines (p=0.009). Gene expression levels of CCNB1, CCNA2, CCNE1 as well as target hallmark scores for “E2F targets”, “G2M checkpoint” and “Mitotic spindle” were significantly higher in TNBC vs other breast cancer cell lines. Oral treatment with CID-016 resulted in tumor regression in at least one triple-negative breast cancer PDX model and was well tolerated with no significant body weight changes. In vivo PD analyses will be assessed after 5 days of treatment with CID-016. Conclusions: The selective cyclin A/B RxL inhibitor CID-016 exhibits potent antitumor activity in TNBC preclinical models, consistent with the proposed mechanism of RB1/E2F pathway dysregulation. Given their compelling characteristics, orally bioavailable RxL inhibitors are advancing as the only first-in-class orally bioavailable dual inhibitors of cyclins A and B into clinical development. Citation Format: Mariana Paes Dias, Li-Fen Liu, Bernard Levin, Cristina Molina, Marta Guzmán, Olga Rodríguez, Evelyn W. Wang, Violeta Serra. Novel orally bioavailable macrocycles that target cyclin A and B elicit antitumor activity in breast cancer patient-derived xenograft models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C04.

Sulindac sulfide as a non-immune suppressive γ-secretase modulator to target triple-negative breast cancer.

Triple-negative breast cancer (TNBC) comprises a heterogeneous group of clinically aggressive tumors with high risk of recurrence and metastasis. Current pharmacological treatment options remain largely limited to chemotherapy. Despite promising results, the efficacy of immunotherapy and chemo-immunotherapy in TNBC remains limited. There is strong evidence supporting the involvement of Notch signaling in TNBC progression. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Notch inhibitors, including g-secretase inhibitors (GSIs), are quite effective in preclinical models of TNBC. However, the success of GSIs in clinical trials has been limited by their intestinal toxicity and potential for adverse immunological effects, since Notch plays key roles in T-cell activation, including CD8 T-cells in tumors. Our overarching goal is to replace GSIs with agents that lack their systemic toxicity and ideally, do not affect tumor immunity. We identified sulindac sulfide (SS), the active metabolite of FDA-approved NSAID sulindac, as a potential candidate to replace GSIs. We investigated the pharmacological and immunotherapeutic properties of SS in TNBC models in vitro, ex-vivo and in vivo. We confirmed that SS, a known γ-secretase modulator (GSM), inhibits Notch1 cleavage in TNBC cells. SS significantly inhibited mammosphere growth in all human and murine TNBC models tested. In a transplantable mouse TNBC tumor model (C0321), SS had remarkable single-agent anti-tumor activity and eliminated Notch1 protein expression in tumors. Importantly, SS did not inhibit Notch cleavage in T- cells, and the anti-tumor effects of SS were significantly enhanced when combined with a-PD1 immunotherapy in our TNBC organoids and in vivo. Our data support further investigation of SS for the treatment of TNBC, in conjunction with chemo- or -chemo-immunotherapy. Repurposing an FDA-approved, safe agent for the treatment of TNBC may be a cost-effective, rapidly deployable therapeutic option for a patient population in need of more effective therapies.

Combined PARP and WEE1 inhibition triggers anti-tumor immune response in BRCA1/2 wildtype triple-negative breast cancer

Novel therapeutic strategies that can effectively combine with immunotherapies are needed in the treatment of triple-negative breast cancer (TNBC). We demonstrate that combined PARP and WEE1 inhibition are synergistic in controlling tumour growth in BRCA1/2 wild-type TNBC preclinical models. The PARP inhibitor (PARPi) olaparib combined with the WEE1 inhibitor (WEE1i) adavosertib triggered increases in anti-tumour immune responses, including STING pathway activation. Combinations with a STING agonist resulted in further improved durable tumour regression and significant improvements in survival outcomes in murine tumour models of BRCA1/2 wild-type TNBC. In addition, we have identified baseline tumour-infiltrating lymphocyte (TIL) levels as a potential predictive biomarker of response to PARPi, WEE1i and immunotherapies in BRCA1/2 wild-type TNBC.

PI3K inhibition circumvents resistance to SHP2 blockade in metastatic triple-negative breast cancer

The protein tyrosine phosphatase SHP2 activates oncogenic pathways downstream of most receptor tyrosine kinases (RTK) and has been implicated in various cancer types, including the highly aggressive subtype of triple-negative breast cancer (TNBC). Although allosteric inhibitors of SHP2 have been developed and are currently being evaluated in clinical trials, neither the mechanisms of the resistance to these agents, nor the means to circumvent such resistance have been clearly defined. The PI3K signaling pathway is also hyperactivated in breast cancer and contributes to resistance to anticancer therapies. When PI3K is inhibited, resistance also develops for example via activation of RTKs. We therefore assessed the effect of targeting PI3K and SHP2 alone or in combination in preclinical models of metastatic TNBC. In addition to the beneficial inhibitory effects of SHP2 alone, dual PI3K/SHP2 treatment decreased primary tumor growth synergistically, blocked the formation of lung metastases, and increased survival in preclinical models. Mechanistically, transcriptome and phospho-proteome analyses revealed that resistance to SHP2 inhibition is mediated by PDGFRβ-evoked activation of PI3K signaling. Altogether, our data provide a rationale for co-targeting of SHP2 and PI3K in metastatic TNBC.

The ARETHA study: A phase 2 randomized control trial of eribulin with evexomostat (SDX-7320) or placebo for patients with metastatic triple-negative breast cancer (TNBC) and metabolic dysfunction.

TPS1131 Background: The prognosis for advanced/metastatic TNBC remains poor and novel therapeutic strategies are urgently needed. Insulin resistance and obesity contribute to TNBC progression and are independent predictors of worse survival. Methionine aminopeptidase 2 (MetAP2/p67) is overexpressed in many tumor types including breast. MetAP2 inhibitors exhibit broad anti-tumor activity and attenuate the effects of metabolic dysfunction on tumor growth. Evexomostat (SDX-7320) is a second-generation MetAP2 inhibitor that improved insulin sensitivity, reduced fat mass, and normalized adipokine levels in preclinical models of obesity and reduced tumor growth in TNBC preclinical models. In phase 1 trials, evexomostat monotherapy partially restored insulin sensitivity, reduced adipokines, and showed anti-metastatic effects in patients with advanced solid tumors. The goal of this phase 2 study is to test whether evexomostat prevents worsening of insulin resistance and augments tumor response in patients with metastatic TNBC and metabolic dysfunction in combination with eribulin chemotherapy, a standard of care treatment option after prior anthracycline and taxane exposure. Methods: This is a single-center phase 2 randomized control trial of evexomostat, a MetAP2 inhibitor, and eribulin chemotherapy. Key eligibility criteria include metastatic TNBC, measurable disease or ≥1 predominantly lytic bone lesion, baseline hemoglobin A1c >5.5% and/or BMI ≥30 kg/m2, and ≤2 prior lines of therapy in the advanced/metastatic setting. Patients with uncontrolled or insulin-dependent type II diabetes, or who require combination antihyperglycemic therapy are excluded. During the safety run-in period, 15 patients will be assigned to receive evexomostat 49 mg/m2 Q2 weeks and eribulin 1.4 mg/m2 on days 1 & 8 of a 21-day cycle. Upon safety confirmation, an additional 40 patients will be randomized 2:1 to receive evexomostat or placebo with eribulin. The primary endpoint is metabolic efficacy assessed by change in Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) score. Secondary endpoints include objective response rate, progression-free survival, safety and tolerability, patient-reported outcomes, and changes in metabolic markers and body composition. Historically, HOMA-IR scores double during chemotherapy, and this trial will test whether evexomostat attenuates the expected HOMA-IR rise. This trial will have >90% power to detect a difference between a 1.5-fold change in HOMA-IR in the control arm vs. no change in the evexomostat arm while controlling the Type I error at 5%. As of Feb 2022, 3 patients are enrolled; total accrual of 61 patients is planned with a goal of 55 evaluable patients. Clinical trial information: NCT05570253 .

Abstract 5483: Combined inhibition of CDK4/6 and AKT is highly active against the luminal androgen receptor (LAR) subtype of triple negative breast cancer (TNBC)

Abstract The LAR subtype of TNBC is enriched for targetable biomarkers, including androgen receptor (AR) expression, PIK3CA mutations, and intact Rb. The purpose of this study was to investigate the most effective combinations of inhibitors of CDK4/6 (palbociclib), AR (enzalutamide), and PI3K-AKT (alpelisib/capivasertib) against preclinical models of LAR TNBC. MDA-MB-453 and MFM-223 (both AR positive/Rb-intact/PIK3CA-mutant) LAR TNBC cells were treated with each inhibitor alone or in different combinations. Drug sensitivity was measured as growth in 2D, colony formation, and CellTiterGlo viability. Synergistic drug effects were expressed as the combination index (CI) calculated by CompuSyn methods. Activation of cell cycle and PI3K-AKT signaling molecules in response to different inhibitors or siRNAs was measured by immunoblot analysis. An androgen response element (ARE) luciferase reporter assay was used to evaluate AR transcriptional activity. MDA-MB-453 and MFM-223 cells were sensitive to single-agent palbociclib, alpelisib, or capivasertib (IC50 ~500 nM) but not enzalutamide (IC50 15-25 µM). Palbociclib combined with either the PI3Kαi alpelisib or the AKTi capivasertib synergistically inhibited growth of both cell lines (CI values, 0.07-0.86). AR transcriptional reporter activity was not altered by any of these treatments. Treatment with palbociclib monotherapy (24 h) suppressed p-Rb and increased phosphorylation of AKTS473 and its substrates FOXO3A, GSK3β, and PRAS40, suggesting PI3K/AKT signaling mediates an adaptive response to CDK4/6 blockade. In MDA-MB-453 cells, palbociclib-induced phosphorylation of AKT substrates was suppressed by treatment with capivasertib but not with alpelisib alone or with the PI3Kβ/δ inhibitor AZD8186, suggesting that the compensatory activation of AKT was due to a mechanism independent of the PI3K isozymes. We next used RICTOR siRNAs to block mTORC2-mediated phosphorylation AKTS473 after palbociclib treatment. Gene silencing of RICTOR reduced levels of the mTORC2 substrate p-SGK1S422 but did not block phosphorylation of AKTS473 and its substrates in palbociclib-treated cells, suggesting that this adaptative response was mTORC2-independent. Mean CI values showed that the combination of palbociclib/capivasertib was clearly more synergistic than palbociclib/alpelisib in both cells lines (mean CI, 0.29 vs. 0.78). Currently, we are comparing both combinations in mice bearing established MDA-MB-453 xenografts. Further investigation of the mechanisms of adaptive activation of AKT upon blockade of CDK4/6 in TNBC cells are in progress. In sum, addition of an AKT inhibitor, but not PI3K inhibitors, to palbociclib suppressed the rebound activation of AKT following treatment with the CDK4/6i, supporting a testable therapeutic strategy in LAR TNBC with intact Rb. Citation Format: Maria del Rosario Chica-Parrado, Gun Min Kin, Chang-Ching Lin, Kyung-min Lee, Fabiana Napolitano, Dan Ye, Emmanuel Bikorimana, Saurabh Mendirata, Ariella Hanker, Carlos L. Arteaga. Combined inhibition of CDK4/6 and AKT is highly active against the luminal androgen receptor (LAR) subtype of triple negative breast cancer (TNBC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5483.

Subtyping-based platform guides precision medicine for heavily pretreated metastatic triple-negative breast cancer: The FUTURE phase II umbrella clinical trial

Triple-negative breast cancer (TNBC) is a heterogeneous disease and lacks effective treatment. Our previous study classified TNBCs into four subtypes with putative therapeutic targets. Here, we report the final results of FUTURE, a phase II umbrella trial designed to explore whether the subtyping-based strategy may improve the outcomes in metastatic TNBC patients. A total of 141 patients with a median of three previous lines of therapies in the metastatic setting were enrolled in seven parallel arms. Confirmed objective responses were achieved in 42 patients (29.8%; 95% confidence interval [CI], 22.4–38.1). The median values of progression-free survival and overall survival were 3.4 (95% CI: 2.7–4.2) and 10.7 (95% CI: 9.1–12.3) months, respectively. Given Bayesian predictive probability, efficacy boundaries were achieved in four arms. Furthermore, integrated genomic and clinicopathological profiling illustrated associations of clinical and genomic parameters with treatment efficacy, and the efficacy of novel antibody–drug conjugates was explored in preclinical TNBC models of subtypes for which treatment was futile. In general, the FUTURE strategy recruits patients efficiently and provides promising efficacy with manageable toxicities, outlining a direction for further clinical exploration.

Abstract OT2-17-02: The ARETHA Study: A phase 2 randomized control trial of Eribulin with Evexomostat (SDX-7320) or placebo for patients with metastatic triple-negative breast cancer and metabolic dysfunction

Abstract Background: The prognosis for advanced/metastatic triple negative breast cancer (TNBC) remains poor and novel therapeutic strategies are urgently needed to improve outcomes. Insulin resistance and obesity contribute to cancer progression and are independent predictors of worse survival after TNBC diagnosis. Chemotherapy worsens insulin resistance, body mass index, and multiple other metabolic parameters which can paradoxically limit treatment efficacy and survival. Methionine aminopeptidase 2 (MetAP2/p67) is overexpressed in many tumor types and MetAP2 inhibitors exhibit broad anti-tumor activity. Additionally, MetAP2 inhibitors improve insulin resistance, reduce adiposity, and normalize levels of adipokines, attenuating the effects of metabolic dysfunction on tumor growth. Evexomostat (SDX-7320) is a second-generation MetAP2 inhibitor designed to improve drug-like properties and minimize central nervous system toxicities. Evexomostat improved insulin sensitivity, reduced fat mass and normalized adipokine levels in preclinical models of obesity, and reduced tumor growth in preclinical models of TNBC. In phase 1 trials, MetAP2 inhibition combined with chemotherapy was safe and well tolerated, while evexomostat monotherapy partially restored insulin sensitivity, reduced angiogenic factors as well as adipokines and showed anti-metastatic effects in patients with advanced solid tumors. The goal of this phase 2 study is to test whether evexomostat in combination with eribulin chemotherapy prevents worsening of insulin resistance and augments tumor response in patients with metastatic TNBC and concomitant metabolic dysfunction. Methods: This is a single-center, placebo-controlled phase 2 randomized control trial of evexomostat, a MetAP2 inhibitor, in combination with eribulin chemotherapy. Eligible patients must have histologically confirmed metastatic TNBC, measurable disease or ≥1 predominantly lytic bone lesion, baseline metabolic dysfunction defined as hemoglobin A1c >5.5% and/or BMI ≥30 kg/m2, have received ≤2 prior lines of therapy in the advanced/metastatic setting, have ECOG performance status ≤1, and adequate organ function. Patients with uncontrolled or insulin-dependent type II diabetes, or who require combination antihyperglycemic therapy are excluded. During the safety run-in period, 15 patients will be assigned to receive evexomostat 49 mg/m2 every 2 weeks in combination with eribulin 1.4 mg/m2 administered on days 1 and 8 of a 21-day cycle. Upon safety confirmation, an additional 40 patients will be randomized 2:1 to receive evexomostat or placebo in combination with eribulin. The primary endpoint is metabolic efficacy assessed by change in Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) score. Secondary endpoints include objective response rate, progression-free survival, duration of response, safety and tolerability, patient-reported outcomes, changes in metabolic markers, and changes in body composition parameters. Historically, HOMA-IR scores double during chemotherapy, and this trial will test whether evexomostat will attenuate the expected rise in HOMA-IR. Specifically, the trial will have >90% power to detect a difference between a 1.5-fold change in HOMA-IR in the control arm versus no change in the investigational arm while controlling the Type I error at 5%. This trial opened to accrual in July 2022. A total accrual of 61 patients is planned with a goal of 55 evaluable patients. For any inquiries/questions, please contact Sherry Shen at shens1@mskcc.org. Clinical trial registry number: pending Funding, study drug evexomostat and other support provided by: SynDevRx, Inc. Citation Format: Sherry Shen, Anna Whalen, Mark E. Robson, Larry Norton, Tiffany A. Traina, Neil M. Iyengar. The ARETHA Study: A phase 2 randomized control trial of Eribulin with Evexomostat (SDX-7320) or placebo for patients with metastatic triple-negative breast cancer and metabolic dysfunction [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-17-02.

Abstract ED9-3: Effects of age on the tumor microenvironment

Abstract Dr. McAllister’s translational research group studies breast cancer as a systemic disease with an emphasis on the role of immune fitness in disease control and response to therapy. In this education session, Dr. McAllister will introduce the concept of “immunological age”. She will discuss her team’s ongoing efforts to understand how age sets the stage for triple-negative breast cancer (TNBC) progression and response to immune checkpoint blockade therapy. Her team was the first to report that bone marrow derived immune cells from 8–10-week-old young mice promote more aggressive TNBC growth when transferred to 12–15-month-old aged mice (Marsh, et al., Cancer Research, 2016). They subsequently identified cellular and molecular mechanisms that promote aggressive disease, as typically observed in younger individuals. Likewise, her group was the first to report that immune checkpoint blockade therapy is less effective in older mice with TNBC and discovered a treatment strategy that improved outcomes in pre-clinical TNBC models (Sceneay, et al., Cancer Discovery, 2019). The same tumor cellular and molecular pathways that stratified by age in their pre-clinical models also defined age-stratified differences between younger (under 40) and older (over 65) TNBC patients in the METABRIC dataset. She will present her team’s new work on functional aspects of immunological age in TNBC and the ongoing efforts of the Older Women with Breast Cancer Research and Treatment Team at the Dana-Farber/Brigham Cancer Center. Ultimately, she and her team wish to eliminate age- and race-based disparities in breast cancer outcomes. Citation Format: Sandra McAllister. Effects of age on the tumor microenvironment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr ED9-3.

Abstract 2639: KB-0742 is active in preclinical MYC high models of TNBC, ovarian, and DLBCL cancers

Abstract MYC is an early-response gene downstream of many receptor complexes, and a component that promotes several pro-growth signal transduction pathways. As a result, MYC expression is highly regulated, but once dysregulated in cancer, MYC promotes tumorigenesis via activation of cell growth, proliferation, and angiogenesis. CDK9 is both an upstream and downstream cofactor of MYC. CDK9 promotes MYC expression through its recruitment to super enhancers at the MYC amplicon and acts as a cofactor to drive gene expression programs. Previously, we profiled pan-cancer sensitivity to KB-0742, a potent, selective, and orally bioavailable small-molecule inhibitor of CDK9, and identified MYC-amplified cancers as being especially sensitive to KB-0742. To better understand the connection between MYC and KB-0742 sensitivity, 11 different tumor types were evaluated preclinically using immortalized cell lines, patient-derived cell lines (PDCs), patient-derived organoids (PDOs), and patient-derived xenografts (PDXs). The 11 indications included head and neck, esophageal, gastric, liver, non-small cell lung cancer (NSCLC), pancreatic, ovarian, triple-negative breast cancer (TNBC), prostate, colon, and lymphoma. For 5 of the indications (head and neck, esophageal, gastric, liver, and colon), little activity was observed in cell lines and in vivo with weak correlations to MYC amplification. TNBC, ovarian cancer, and lymphoma showed good responses to KB-0742 treatment. For TNBC and ovarian, immortalized and patient-derived cell lines indicated lower IC50 values with increased MYC amplification or expression, but the results were not statistically significant. However, in vivo assessments using 15 PDX models showed good correlation of tumor growth inhibition (TGI) and MYC amplification/expression, with some tumor regressions observed in several ovarian models. Two PDO models of TNBC with different treatment histories were also treated with KB-0742 and compared to 4 standard-of-care (SOC) compounds. KB-0742 showed much greater activity in the models, with maximal inhibition rates of 100% and 89%. Cell-line screens of blood cancer-derived cell lines showed that lymphocyte-derived cell lines were the most sensitive to KB-0742 as compared to all other cancer types. We tested several PDX models for response to our compound. Treatment with KB-0742 resulted in TGIs of over 50% in several lymphoma models, including a TGI of 56% in 1 model of double-hit diffuse large B-cell lymphoma. These data support the continual development of KB-0742, which is currently being tested in a phase 1/2 clinical trial (NCT04718675). The dose-escalation phase is opened to relapsed or refractory solid tumors or non-Hodgkin lymphoma. Once a recommend phase 2 dose is determined, we plan to open dedicated expansion arms enrolling patients with MYC over-expression/amplification within the tumor types supported by these studies. Citation Format: Melinda A. Day, Douglas C. Saffran, Tressa Hood, Nikolaus Obholzer, Akanksha Pandey, Charles Y. Lin, Pavan Kumar, Jorge DiMartino. KB-0742 is active in preclinical MYC high models of TNBC, ovarian, and DLBCL cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2639.