Resistant Triple Negative Breast Cancer Research Articles

Proteomic Dynamics of Breast Cancer Cell Lines Identifies Potential Therapeutic Protein Targets

Treatment and relevant targets for breast cancer (BC) remain limited, especially for triple-negative BC (TNBC). We identified 6091 proteins of 76 human BC cell lines using data-independent acquisition (DIA). Integrating our proteomic findings with prior multi-omics datasets, we found that including proteomics data improved drug sensitivity predictions and provided insights into the mechanisms of action. We subsequently profiled the proteomic changes in nine cell lines (five TNBC and four non-TNBC) treated with EGFR/AKT/mTOR inhibitors. In TNBC, metabolism pathways were dysregulated after EGFR/mTOR inhibitor treatment, while RNA modification and cell cycle pathways were affected by AKT inhibitor. This systematic multi-omics and in-depth analysis of the proteome of BC cells can help prioritize potential therapeutic targets and provide insights into adaptive resistance in TNBC.

Tumor sensitization to PARP inhibitors by DNA damaging synthetically lethal acylfulvenes.

e15123 Background: 20-30% of solid tumors (ovarian, breast, pancreatic, prostate cancers) harbor some level of deficiency in DNA Damage Repair (DDR) pathways. Such cancers become dependent on alternative DDR pathways to maintain genome integrity, making them selectively vulnerable to DNA damaging agents. The concept of synthetic lethality has been leveraged to treat subsets of such tumors. Clinically, PARP inhibitors (PARPi) have been successful in Homologous Recombination Deficiency (HRD) cancers. LP-100 and LP-184 are acylfulvene (AF) class small molecule prodrugs that are metabolized to active compounds by prostaglandin reductase 1 (PTGR1) that is often elevated in solid tumors. AF-induced DNA lesions are likely selectively processed by repair pathways including BRCA1/2 mediated Homologous Recombination (HR). Methods: Cell Titer Glo based 10 day cell viability assays were used to generate drug IC50 values in vitro. Bliss synergy scores were computed with scores ≥ 10 reflecting synergy. Drug-induced DNA double strand breaks (DSB) were detected and quantified with STRIDE (SensiTive Recognition of Individual DNA Ends) assay in cancer cells. A subcutaneous tumor xenograft model was used to determine tumor volumes in control and drug treated mice. Results: In a BRCA2 mutant ovarian cancer cell line OVCAR3, LP-184 and olaparib co-treatment in vitro showed synergistic effects (Bliss score 10). Similarly in a BRCA2 mutant prostate cancer cell line 22Rv1, LP-100 combination with rucaparib was synergistic (Bliss score 18). 400 nM LP-184 showed a time dependent increase in DNA DSB signals in vitro, 1.7X more so in BRCA2 mutant vs wild type (WT) DLD-1 isogenic colon cancer cell lines up to 24 hours. To validate the hypothesis that AF-induced DSBs remain unresolved if exposed to and further synergize with PARPi in vivo, we tested LP-100 plus olaparib in a DU145 (BRCA2 mutant) prostate cancer xenograft model. LP-100 + olaparib treatment resulted in sustained, durable tumor regression indicating in vivo synergy whereas either agent alone did not produce tumor shrinkage. Day 42 tumor growth inhibition was 66% for 2.5 mg/kg LP-100 alone, 34% for 40 mg/kg olaparib alone and 108% for the combination. Moreover, LP-184 treatment resulted in early onset and complete tumor regression in 7 PDX models of PARPi resistant triple negative breast cancers (TNBC) harboring BRCA1/2 loss whereas olaparib/ niraparib remained entirely ineffective in these. Conclusions: AF response is influenced by tumor DNA repair competence. LP-100 and LP-184 are synthetically lethal resulting from their abilities to cause unresolvable DNA damage where tumor cells express high PTGR1 and are deficient in the HR pathway. LP-184 single agent was superior to PARPi in TNBC and LP-100 combination made an olaparib resistant tumor highly sensitive, thereby facilitating a path to extend the opportunities for both AFs and PARPis and enhance the clinical utilization of PARPi.

PRMT5 reduces immunotherapy efficacy in triple-negative breast cancer by methylating KEAP1 and inhibiting ferroptosis

BackgroundAs an emerging treatment strategy for triple-negative breast cancer (TNBC), immunotherapy acts in part by inducing ferroptosis. Recent studies have shown that protein arginine methyltransferase 5 (PRMT5) has distinct roles...

The Suppressive Effect of Rebastinib on Triple-negative Breast Cancer Tumors Involves Multiple Mechanisms of Action.

High resistance of triple-negative breast cancer has prompted scientists to look for new targets susceptible to treatment. CDK16 has been suggested as a promising target whose inhibition can lead to tumor growth suppression. Rebastinib, a potent inhibitor of CDK16, has been reported to exhibit anti-tumor activity both in vitro and in vivo. The anticancer activity of rebastinib was studied in vitro using cell proliferation, cell cycle arrest and cell apoptosis assays and in vivo in xenograft tumor models using MDA-MB-231 and MDA-MB-468-derived tumors. The safety and drug-like properties of rebastinib were assessed using a panel of Absorption, Distribution, Metabolism, and Excretion (ADME) assays, Ames tests, human Ether-a-go-go Related Gene (hERG) experiments and pharmacokinetic studies in mice and rats. Rebastinib demonstrates antitumor activity against breast cancer both in vitro and in vivo. However, the response of the tumor strongly depends on the type of triple-negative breast cancer. Rebastinib-induced cell cycle arrest was observed in G0/G1 phase suggesting a more complex mechanism than just CDK16 inhibition. ADME and PK studies confirmed the drug-like properties and reasonable safety of rebastinib. Our studies confirmed rebastinib to be a promising drug candidate for breast cancer treatment with high oral bioavailability and reasonable safety. Our data suggest that the mechanism of action of rebastinib is not limited to CDK16 inhibition but also involves other pathways. This does not diminish the importance of rebastinib as a drug candidate, but reveals the presence of several mechanisms, suggesting a wider scope of possible applications.

Everolimus combined with PD-1 blockade inhibits progression of triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Due to rapid progression and a lack of targetable receptors, TNBC is exceptionally difficult to treat. Available treatment options are nonspecific cytotoxic agents, which have had modest success; thus, there is a need for novel therapies for TNBC. The mammalian/mechanistic target of rapamycin (mTOR) signaling pathway is aberrantly activated in TNBC, and this pathway has been shown to promote cancer cell survival and chemoresistance. As such, mTOR inhibition has been considered a potential therapeutic strategy for TNBC. The mTOR inhibitor everolimus (EVE) has been approved for the treatment of estrogen positive breast cancer; however, its efficacy in TNBC is still undetermined. In this study, we evaluated the effects of EVE monotherapy and the mechanism of EVE resistance in the 4T1 model of TNBC. Whereas EVE monotherapy inhibited mTOR signaling activity, it did not attenuate tumor progression. Additionally, tumors from EVE-treated mice had abnormal vasculature characterized by disorganized architecture and hyperpermeability. We also found that treatment with EVE increased PD-L1 expression in intratumoral vascular endothelial cells, and this increase in endothelial cell-associated PD-L1 corresponded to reduced CD8 + T cell tumor infiltration. Importantly, combination treatment with anti-PD-1 antibody and EVE normalized the tumor vasculature, rescued CD8 + T cell tumor infiltration, and reduced tumor growth. Taken together, our findings improve our current understanding of mechanisms underlying mTOR inhibition resistance in TNBC and identify a novel combination treatment strategy in the treatment of mTOR resistant tumors.

Monoacylglycerol Lipase Inhibitor JJKK048 Ameliorates ABCG2 Transporter-Mediated Regorafenib Resistance Induced by Hypoxia in Triple Negative Breast Cancer Cells

Triple negative breast cancer (TNBC) is among the most aggressive and deadly cancer subtypes. Intra-tumoral hypoxia is associated with aggressiveness and drug resistance in TNBC. One of the underlying mechanisms of hypoxia-induced drug resistance is the elevated expression of efflux transporters such as breast cancer resistant protein (ABCG2). In the present study, we investigated the possibility of ameliorating ABCG2-mediated drug resistance in hypoxic TNBC cells by monoacylglycerol lipase (MAGL) inhibition and the consequent downregulation of ABCG2 expression.The effect of MAGL inhibition on ABCG2 expression, function, and efficacy of regorafenib, an ABCG2 substrate was investigated in cobalt dichloride (CoCl2) induced pseudohypoxic TNBC (MDA-MB-231) cells, using quantitative targeted absolute proteomics, qRT-PCR, anti-cancer drug accumulation in the cells, cell invasiveness and resazurin-based cell viability assays.Our results showed that hypoxia-induced ABCG2 expression led to low regorafenib intracellular concentrations, reduced the anti-invasiveness efficacy, and elevated half maximal inhibitory concentration (IC50) of regorafenib in vitro MDA-MB-231 cells. MAGL inhibitor, JJKK048, reduced ABCG2 expression, increased regorafenib cell accumulation, which led to higher regorafenib efficacy.In conclusion, hypoxia-induced regorafenib resistance due to ABCG2 over-expression in TNBC cells can be ameliorated by MAGL inhibition.

Activated T cell-derived exosomes for targeted delivery of AXL-siRNA loaded paclitaxel-poly-L-lysine prodrug to overcome drug resistance in triple-negative breast cancer

Triple-negative breast cancer (TNBC) still lacks effective treatment options due to tumor heterogeneity and immunosuppressive microenvironment caused chemoimmunotherapy therapy resistance. Here, the programmed death ligand-1 (PD-L1) and AXL protein are observed to be overexpressed in the paclitaxel (PTX)-resistant TNBC cell lines. AXL overexpression mediates PTX resistance and immune escape by enhancing the epithelial − mesenchymal transition (EMT) process and tumor cell stemness. Hence, an AXL-siRNA (siAXL) loaded glutathione (GSH)-responsive PTX-poly-L-lysine (PTX-PLL; PP) prodrug micelle encapsulated within activated T cell-derived exosome delivery system (PP/siAXL@EXO) is developed to overcome the above obstacles. At the tumor site, exosomal PD-1 attenuates PD-L1-induced immune suppression and enhances cellular uptake. In tumor cells, the first released siAXL overcomes PTX resistance by reversing EMT and inhibits T cell exhaustion, and subsequent high GSH-triggered disulfide bond cleavage promotes GSH depletion and PTX release to kill tumor cells. Moreover, AXL knockdown and PTX-PLL prodrugs synergistically induce immunogenic tumor cell death to enhance immune-killing effect mediated by exosomal PD-1, forming a positive feedback anti-tumor immune cycle. PP/siAXL@EXO overcomes TNBC heterogeneity and significantly inhibit PTX-resistance tumor growth and metastasis, and induce protective immune memory with excellent biosafety. Collectively, PP/siAXL@EXO platform, realizing tumor-specific spatiotemporally synchronized gene-chemoimmunotherapy, provides a potential therapeutic strategy for TNBC.

Triumphs and challenges in exploiting poly(ADP-ribose) polymerase inhibition to combat triple-negative breast cancer.

Poly(ADP-ribose) polymerase 1 (PARP1) regulates a myriad of DNA repair mechanisms to preserve genomic integrity following DNA damage. PARP inhibitors (PARPi) confer synthetic lethality in malignancies with a deficiency in the homologous recombination (HR) pathway. Patients with triple-negative breast cancer (TNBC) fail to respond to most targeted therapies because their tumors lack expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Certain patients with TNBC harbor mutations in HR mediators such as breast cancer susceptibility gene 1 (BRCA1) and breast cancer susceptibility gene 2 (BRCA2), enabling them to respond to PARPi. PARPi exploits the synthetic lethality of BRCA-mutant cells. However, de novo and acquired PARPi resistance frequently ensue. In this review, we discuss the roles of PARP in mediating DNA repair processes in breast epithelial cells, mechanisms of PARPi resistance in TNBC, and recent advances in the development of agents designed to overcome PARPi resistance in TNBC.

Pd(II) and Pt(II) Trinuclear Chelates with Spermidine: Selective Anticancer Activity towards TNBC-Sensitive and -Resistant to Cisplatin.

Triple-negative breast cancer (TNBC) is one of the most aggressive forms of breast cancer and constitutes 10-20% of all breast cancer cases. Even though platinum-based drugs such as cisplatin and carboplatin are effective in TNBC patients, their toxicity and development of cancer drug resistance often hamper their clinical use. Hence, novel drug entities with improved tolerability and selectivity profiles, as well as the ability to surpass resistance, are needed. The current study focuses on Pd(II) and Pt(II) trinuclear chelates with spermidine (Pd3Spd2 and Pt3Spd2) for evaluating their antineoplastic activity having been assessed towards (i) cisplatin-resistant TNBC cells (MDA-MB-231/R), (ii) cisplatin-sensitive TNBC cells (MDA-MB-231) and (iii) non-cancerous human breast cells (MCF-12A, to assess the cancer selectivity/selectivity index). Additionally, the complexes' ability to overcome acquired resistance (resistance index) was determined. This study revealed that Pd3Spd2 activity greatly exceeds that displayed by its Pt analog. In addition, Pd3Spd2 evidenced a similar antiproliferative activity in both sensitive and resistant TNBC cells (IC50 values 4.65-8.99 µM and 9.24-13.34 µM, respectively), with a resistance index lower than 2.3. Moreover, this Pd compound showed a promising selectivity index ratio: >6.28 for MDA-MB-231 cells and >4.59 for MDA-MB-231/R cells. Altogether, the data presently gathered reveal Pd3Spd2 as a new, promising metal-based anticancer agent, which should be further explored for the treatment of TNBC and its cisplatin-resistant forms.

Abstract 1762: MALT1 is activated by doxorubicin and mediates therapy resistance in triple-negative breast cancer

Abstract Background: Breast cancer is the most commonly diagnosed malignancy in American women. The triple-negative breast cancer (TNBC) subtype has among the worst prognosis due to high rates of recurrence and metastasis. Since TNBC lacks targetable receptor proteins, treatment relies upon non-specific chemotherapy, which can become ineffective upon onset of resistance. One potential driver of TNBC treatment resistance is MALT1, the effector component of the CARMA-BCL10-MALT1 signaling complex, which activates NF-κB in multiple cancer cell types, including TNBC. Notably, breast cancer cells demonstrate increased sensitivity to chemotherapies such as doxorubicin and cisplatin when MALT1 is depleted. NF-κB activation has been suggested to promote DNA repair and the MALT1-NF-κB inducible factor, IL-6 has been shown to repress the immunogenic effects of doxorubicin. Hence, we hypothesize that MALT1 is a pharmaceutically targetable driver of TNBC treatment resistance. Mechanistically, we hypothesize that MALT1 promotes chemotherapy resistance by enhancing DNA repair and suppressing immunogenic cell death. Methods/Results: We analyzed RNAseq and proteomic data from TCGA and CPTAC, respectively, and found that MALT1 is highly expressed in basal breast cancer (a subtype largely composed of TNBC) and its expression level in this context is associated with reduced pathological complete response and survival. With the purpose of evaluating the effect of MALT1 protease inhibition on chemotherapy sensitivity, we first sought to identify TNBC cell lines that were most resistant to doxorubicin using GDSC and CTRP databases. Results indicated that MDA-MB-231, BT20 and HCC1143 were highly resistant. We then performed western blots to assess MALT1 expression and CellTiter-Glo assays to determine the doxorubicin IC50s for these cell lines. Results indicate that MALT1 expression correlates with the degree of doxorubicin resistance among these cell lines. To determine if MALT1 blockade, via siRNA-knockdown or MALT1 protease inhibitor treatment (JNJ-67856633 or MLT-748), increased doxorubicin sensitivity, CellTiter-Glo and Incucyte Caspase-3/7 assays were performed. Findings indicated that MALT1 blockade results in decreased cell viability and increased apoptosis in response to doxorubicin. Mechanistically, MALT1 protease is activated by doxorubicin and MALT1 inhibition increases expression of the immunogenic cell death protein, calreticulin (CRT) in response to doxorubicin. Conclusions and Future Directions: Initial studies suggest that targeting MALT1 enhances TNBC sensitivity to doxorubicin. We will next assess the effect of MALT1 blockade on DNA repair mechanisms in doxorubicin-treated TNBC cells. Additionally, we will test the role of MALT1 in suppressing immunogenic cell death. Through these studies, we hope to inform new approaches for improving treatment response in TNBC. Citation Format: Maria Beecher, Dong Hu, Linda Klei, Jack Little, Linda M. McAllister-Lucas, Peter C. Lucas. MALT1 is activated by doxorubicin and mediates therapy resistance in triple-negative breast cancer [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 1762.

Abstract 2846: The mitochondrial dynamics of breast cancer regulate P-glycoprotein expression

Abstract Multidrug efflux transporter ABCB1 (MDR1) encodes P-glycoprotein (P-gp), a well-known transporter that is believed to contribute to the development of multidrug resistance (MDR). Furthermore, P-gp plays an important role in drug disposition, pharmacokinetics, and drug development. Thus, P-gp transporter expression, function, and modulation of MDR in human cancers have been extensively studied. In spite of significant progress in understanding P-gp, ABCB1 regulation and its impact on clinical trials remain unclear. Through sequential exposure of SUM159PT to paclitaxel, we created a paclitaxel resistant triple negative breast cancer cell line SUM159PT-PAC200. We observed a significant increase in the phosphorylation of the mitochondrial fission protein dynamin related protein kinase 1 (Drp1) at Ser616 and its upstream kinase ERK1/2a of Drp1. Through the use of CRISPR-Cas9 technology, we knocked out the gene encoding Drp1 in PAC200. When Drp1 is genetically abated (PAC200 Drp1-/-), paclitaxel resistance is significantly reversed. It was also observed that clonogenicity under paclitaxel pressure was significantly decreased in both the PAC200/Drp1-/- line and the partial KO Drp1-/+ line, compared to the PAC200 line. Parallelly, a significant increase in the expression of P-gp transporters was also observed in the PAC200 cell line. As a surprise, genetically targeting Drp1 in PAC200 cells (i.e., PAC200 Drp1/KO) reduced P-gp expression to virtually undetectable levels. Until now, there has been no description of how Drp1 and P-gp are related. Furthermore, PAC200 Drp1/KO cells showed reduced proliferation, migration, and invasion potential, while their soft agar clonogenic capacities were almost nonexistent, suggesting that Drp1 plays a key role in breast cancer proliferation and dissemination. It appears that modulating or targeting Drp1 might be a useful strategy for modulating P-gp in the development and maintenance of the cancer's MDR phenotype, proliferation, and invasion. Understanding how mitochondrial dynamics determine the transactivation and coordination of drug-resistant P-gp phenotypes would make Drp1 an attractive molecular target for cancer treatment. Citation Format: David Terrero, Saloni Malla, Dayanidhi Raman, Jayachandra Babu, Amit Tiwari. The mitochondrial dynamics of breast cancer regulate P-glycoprotein expression [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 2846.

Abstract 393: Determinants of birinapant efficacy in triple negative breast cancer

Abstract Background: Triple-Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer with few targeted therapies, limiting patients to harsh systemic treatments and resulting in the lowest 5-year survival rate of all breast cancer subtypes. This survival rate plummets after metastasis to another site, making it crucial to identify new therapeutic options for TNBC patients to prevent recurrence and metastasis. The small molecule therapy birinapant, a secondary mitochondrial activator of caspases (SMAC) mimetic, represents a potential new avenue for TNBC treatment. Birinapant initiates apoptotic signaling through targeting of Inhibitor of Apoptosis (IAP) proteins in the Tumor Necrosis Factor Receptor (TNFR) extrinsic apoptosis signaling pathway, leading to the formation of a death complex, caspase activity, and apoptosis. We have found that approximately 25% of TNBC patient-derived xenograft (PDX) and xenograft-derived organoid (PDxO) models are exquisitely sensitive to birinapant treatment. However, the majority of our TNBC patient-derived lines remain resistant through unknown means. We seek to understand the parameters for birinapant response and the mechanism of inherent resistance in TNBC using our patient-derived TNBC models. Approach and Results: Preliminary Western blot experiments revealed that treatment of birinapant-sensitive and resistant PDX tumors in vivo and PDxOs in vitro with birinapant causes degradation of cIAP1 and cIAP2, but only causes apoptosis in birinapant-sensitive lines. Therefore, there may be a disruption of necessary downstream apoptotic signaling in the birinapant-resistant lines. Through follow-up Western blot experiments we determined that at baseline, birinapant-resistant PDxOs do not lack the TNFR extrinsic apoptosis proteins required for apoptosis signaling after IAP degradation, nor is there an abundance of these proteins in the birinapant-sensitive PDxOs. Furthermore, to determine if TNFR extrinsic apoptosis signaling is impaired in birinapant-resistant PDxOs, we treated birinapant-resistant and -sensitive PDxOs with the TNF-a cytokine, which is known to enhance extrinsic apoptosis signaling in the presence of birinapant. Treatment with TNF-a and birinapant did not induce apoptosis in the birinapant-resistant PDxOs, suggesting that the extrinsic apoptosis signaling pathway may be the culprit of resistance in these patient-derived lines. Discussion: Follow-up experiments will address whether an inability to execute extrinsic apoptosis through other death receptor pathways explains birinapant resistance in these models. This work will allow us to uncover what predisposes TNBC patients to be resistant or sensitive to birinapant, and determine how this may be harnessed therapeutically to improve outcomes for TNBC patients. This project was funded in part with federal funds under HHSN261201500003I and U54CA224076. Citation Format: Elisabeth A. Brown, Chieh-Hsiang Yang, Emilio Cortes-Sanchez, Zhengtao Chu, Jeevan Govindharajulu, Ralph Parchment, James Doroshow, Apurva Srivastava, Alana Welm. Determinants of birinapant efficacy in triple negative breast cancer [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 393.

Abstract 487: ITI-1020, a novel PDE type 1 inhibitor, reduces tumor growth in triple negative breast cancer (TNBC) mouse models

Abstract Breast cancer is the most common cancer in women. Unfortunately, some patients do not benefit from most therapies and may even develop resistance to previously effective treatments leading to disease progression. Therefore, treatment resistance remains a significant challenge and new treatments are needed to overcome this problem. ITI-1020 is a potent and highly selective inhibitor of the phosphodiesterase 1 (PDE1) enzyme. Given the role of PDE1 in the regulation of essential cellular functions, including among others the transition of monocytes to macrophages and potentially the infiltration of macrophages into the tumor microenvironment (TME), we hypothesize that ITI-1020 will alter macrophage infiltration and polarization, as well as improve T cell function and recruitment into the TME, thereby promoting antitumor immunity in murine cancer models. We tested this hypothesis by studying the effect of ITI-1020 given, alone or in combination a PD-1 immune checkpoint inhibitor, on tumor growth, and TME composition of E0771 and 4T1 tumor-bearing mice, two syngeneic models of TNBC. Mice treated with ITI-1020 alone or in combination with anti-PD-1 antibody showed significant inhibition of tumor growth compared to isotype controls in both type of tumors. Flow cytometry analysis of the tumor microenvironment showed a significant shift in the polarization of macrophages toward a more inflammatory phenotype, and/or a reduction in the number of exhausted T cells. Furthermore, scRNAseq analysis of tumors from mice treated with ITI-1020 in monotherapy showed altered expression of genes involved in inflammatory processes and cell proliferation within the tumor microenvironment and in the cancer cells. These results suggest that ITI-1020 promotes antitumor immunity by a novel mechanism (PDE1 inhibition), leading to tumor growth inhibition in E0771 and 4T1 tumor models. Translating these findings to the clinic may provide a new hope for the treatment of refractory or resistant TNBC tumors. Citation Format: Maud Voisin, Aksinija A. Kogan, Stephen Goding, Gretchen L. Snyder, Robert E. Davis. ITI-1020, a novel PDE type 1 inhibitor, reduces tumor growth in triple negative breast cancer (TNBC) mouse models [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 487.

Abstract 2681: Combination therapy of heme inhibitory protein (HeSP2) and Cyclopamine tartrate (CycT) with chemotherapeutic drugs is an effective strategy for treatment of TNBC

Abstract Breast cancer (BC) is the second leading cause of cancer death in women. Among the four main types of BC, triple-negative breast cancer (TNBC) accounts for 10 to 15% of BC. Although most TNBC patients have a poor prognosis, there are subsets of patients with tumors that respond well to chemotherapy. However, the outcome of chemotherapy in TNBC worsens over time due to the high expression of the oxidative phosphorylation (OXPHOS) complex. Likewise, drug resistant TNBC cells depend on OXPHOS; targeting oxidative metabolism and mitochondrial respiration can potentially overcome their drug resistance. Our lab recently engineered a heme-sequestering protein 2 (HeSP2) based on HasA hemophore. HeSP2 can effectively inhibit heme uptake and decrease the OXPHOS levels in cancer cells due to its high affinity for heme. Another agent, cyclopamine tartrate (CycT), previously known as a modulator of hedgehog signaling and mitochondrial respiration, can inhibit heme synthesis and reduce the OXPHOS levels. Therefore, using these heme-targeting agents in combination with chemotherapy could be an effective strategy to increase the therapeutic efficacy in TNBC. Cisplatin and etoposide are two chemotherapeutic drugs that have recently been used for treating TNBC. For this purpose, we examine the combination therapy of our OXPHOS inhibitory agents, HeSP2 and CycT, with cisplatin and etoposide in TNBC. In vitro experiment using three different TNBC cell lines, 4T1-Fluc-Neo, EMT6_Fluc_Puro, and MDA-MBA-231 confirmed the inhibitory effect of both HeSP2 and CycT in cell proliferation and colony formation in combination with or without chemo drugs. Results indicate that combination therapy significantly reduced the proliferation and inhibited colony formation in TNBC cells. As HeSP2 and CycT are therapeutic agents that reduce hypoxia and OXPHOS, they may also delay the emergence of drug resistance in cancer cells treated with chemotherapy. Our results indicate that HeSP2 and CycT in combination with chemotherapy drugs could serve as a promising therapy to diminish the tumorigenic function in TNBC and overcome resistance to chemotherapy. Citation Format: Parinaz Sadat Alemi, Maria del Carmen Chacon Castro, Eranda Berisha, Zakia Akter, Li Zhang. Combination therapy of heme inhibitory protein (HeSP2) and Cyclopamine tartrate (CycT) with chemotherapeutic drugs is an effective strategy for treatment of 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 2681.

Abstract 2774: Moesin-induced adriamycin resistance through IL-6/MSN/STAT3 axis and therapeutic efficacy of atovaquone in chemotherapy resistant triple-negative breast cancer

Abstract Introduction: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a high frequency of chemotherapy resistance. Moesin (MSN) is a cytoskeleton linker discovered as a novel oncogene in our previous proteomics analysis. Here, we evaluated the molecular mechanisms of MSN and how it functions in response to chemotherapy, which remains unclear in TNBC. Method: The 178 in-house TNBC patient cohort and METABRIC data set were enrolled for clinicopathologic analysis. The multiple TNBC four-cell lines were used to establish MSN knock-down and over-expressed in vitro models using a lentiviral system. RNA sequencing was analyzed with MDA-MB-231 parent cell and MDA-MB-231/ADR12 (adriamycin resistance cell line). Invasion and migration assay was done. The drug response based on MSN gene expression was observed by cell viability assay, western blot, and AV/PI flow cytometry. Drug resistance concerning cancer stemness was confirmed with sphere formation and ALDH activity assay. Immunofluorescence and immunoprecipitation were performed. The combination index(CI), which is to define the synergism of combined treatment was calculated with the Compusyn program. Results: The cohort with METABRIC and our in-house clinical data showed concordant findings that overexpressed MSN mRNA and protein expressions have a poor prognosis in TNBC patients. The MSN protein expression in TNBC was increased compared to the protein level in both the normal human tissues and non-TNBC cell lines. Overexpressed MSN induced high cell proliferation activity, invasion, and migration ability. The apoptosis rate (AV+/PI+) along with cleaved PARP and caspase-3 expression was increased in TNBC cell lines with MSN downregulation. Further tests to determine the role of MSN in adriamycin resistance showed an increased number and size of spheres in the MSN overexpressed cell lines. Moreover, the protein and mRNA levels of MSN expression in MDA-MB-231/ADR12 were significantly higher than the level in the matched parental cell line. MSN and phospho-STAT3(T705) were increased by treatment of IL-6 in TNBC cell lines. The direct binding between MSN and STAT3, and the co-localization of MSN and pSTAT3 were consistently confirmed by co-immunoprecipitation and immunofluorescence, which were all activated by IL-6. Blocking the STAT3 pathway with atovaquone restored ADR response in MSN overexpressed tumor cells. CI value on the combined treatment of ADR and atovaquone in the MDA-MB-231/ADR12 cell line showed higher synergetic efficacy than the parental cell line. Conclusion: This research demonstrates the involvement of the MSN in doxorubicin resistance through IL-6/MSN/STAT3 pathway and provides a novel therapeutic candidate for TNBC patients. Citation Format: Cheng Hyun Lee, Da Sol Kim, Soo Young Park, Han Suk Ryu. Moesin-induced adriamycin resistance through IL-6/MSN/STAT3 axis and therapeutic efficacy of atovaquone in chemotherapy resistant triple-negative breast cancer [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 2774.

Cutting the root: the next generation of T cells engagers against cancer stem cells to overcome drug resistance in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most difficult type of breast cancer to treat. TNBC is defined by the lack of expression of three receptors: estrogen receptor (ER); progesterone receptor (PR); and human epidermal growth factor receptor 2 (HER2). Chemotherapy is currently first-line

The WAVE3/β-catenin oncogenic signaling regulates chemoresistance in triple negative breast cancer

BackgroundMetastatic breast cancer is responsible for the death of the majority of breast cancer patients. In fact, metastatic BC is the 2nd leading cause of cancer-related deaths in women in the USA and worldwide. Triple negative breast cancer (TNBC), which lacks expression of hormone receptors (ER-α and PR) and ErbB2/HER2, is especially lethal due to its highly metastatic behavior, propensity to recur rapidly, and for its resistance to standard of care therapies, through mechanisms that remain incompletely understood. WAVE3 has been established as a promoter of TNBC development and metastatic progression. In this study, we investigated the molecular mechanisms whereby WAVE3 promotes therapy-resistance and cancer stemness in TNBC, through the regulation of β-catenin stabilization.MethodsThe Cancer Genome Atlas dataset was used to assess the expression of WAVE3 and β-catenin in breast cancer tumors. Kaplan–Meier Plotter analysis was used to correlate expression of WAVE3 and β-catenin with breast cancer patients’ survival probability. MTT assay was used to quantify cell survival. CRISPR/Cas9-mediated gene editing, 2D and 3D tumorsphere growth and invasion assays, Immunofluorescence, Western blotting, Semi-quantitative and real-time quantitative PCR analyses were applied to study the WAVE3/β-catenin oncogenic signaling in TNBC. Tumor xenograft assays were used to study the role of WAVE3 in mediating chemotherapy resistance of TNBC tumors.ResultsGenetic inactivation of WAVE3 in combination of chemotherapy resulted in inhibition of 2D growth and 3D tumorsphere formation and invasion of TNBC cells in vitro, as well as tumor growth and metastasis in vivo. In addition, while re-expression of phospho-active WAVE3 in the WAVE3-deficient TNBC cells restored the oncogenic activity of WAVE3, re-expression of phospho-mutant WAVE3 did not. Further studies revealed that dual blocking of WAVE3 expression or phosphorylation in combination with chemotherapy treatment inhibited the activity and expression and stabilization of β-catenin. Most importantly, the combination of WAVE3-deficiency or WAVE3-phospho-deficiency and chemotherapy suppressed the oncogenic behavior of chemoresistant TNBC cells, both in vitro and in vivo.ConclusionWe identified a novel WAVE3/β-catenin oncogenic signaling axis that modulates chemoresistance of TNBC. This study suggests that a targeted therapeutic strategy against WAVE3 could be effective for the treatment of chemoresistant TNBC tumors.

Estrogen Receptor β4 Regulates Chemotherapy Resistance and Induces Cancer Stem Cells in Triple Negative Breast Cancer.

Triple Negative Breast Cancer (TNBC) has the worst prognosis among all breast cancers, and survival in patients with recurrence is rarely beyond 12 months due to acquired resistance to chemotherapy, which is the standard of care for these patients. Our hypothesis is that Estrogen Receptor β1 (ERβ1) increases response to chemotherapy but is opposed by ERβ4, which it preferentially dimerizes with. The role of ERβ1 and ERβ4 in influencing chemotherapy sensitivity has never been studied before. CRISPR/CAS9 was used to truncate ERβ1 Ligand Binding Domain (LBD) and knock down the exon unique to ERβ4. We show that the truncated ERβ1 LBD in a variety of mutant p53 TNBC cell lines, where ERβ1 ligand dependent function was inactivated, had increased resistance to Paclitaxel, whereas the ERβ4 knockdown cell line was sensitized to Paclitaxel. We further show that ERβ1 LBD truncation, as well as treatment with ERβ1 antagonist 2-phenyl-3-(4-hydroxyphenyl)-5,7-bis(trifluoromethyl)-pyrazolo[1,5-a] pyrimidine (PHTPP), leads to increase in the drug efflux transporters. Hypoxia Inducible Factors (HIFs) activate factors involved in pluripotency and regulate the stem cell phenotype, both in normal and cancer cells. Here we show that the ERβ1 and ERβ4 regulate these stem cell markers like SOX2, OCT4, and Nanog in an opposing manner; and we further show that this regulation is mediated by HIFs. We show the increase of cancer cell stemness due to ERβ1 LBD truncation is attenuated when HIF1/2α is knocked down by siRNA. Finally, we show an increase in the breast cancer stem cell population due to ERβ1 antagonist using both ALDEFLUORTM and SOX2/OCT4 response element (SORE6) reporters in SUM159 and MDA-MB-231 cell lines. Since most TNBC cancers are ERβ4 positive, while only a small proportion of TNBC patients are ERβ1 positive, we believe that simultaneous activation of ERβ1 with agonists and inactivation of ERβ4, in combination with paclitaxel, can be more efficacious and yield better outcome for chemotherapy resistant TNBC patients.

Tumor targeted combination therapeutic system for the effective treatment of drug resistant triple negative breast cancer

Breast cancer has become the malignant tumor with the largest incidence, especially the drug resistant triple negative breast cancer (TNBC). The combination therapeutic system can play a better role in resisting drug resistant TNBC. In this study, dopamine and tumor targeted folic acid modified dopamine were synthesized as carrier materials to construct melanin-like tumor targeted combination therapeutic system. The optimized nanoparticles of CPT/Fe@PDA-FA10 with efficient loading of camptothecin and iron was achieved, which showed tumor targeted delivery ability, pH sensitive controlled release, effective photothermal conversion performance and excellent anti-tumor efficacy in vitro and in vivo. CPT/Fe@PDA-FA10 plus laser could significantly kill the drug resistant tumor cells, inhibit the growth of the orthotopic drug resistant triple negative breast cancer through apoptosis/ferroptosis/photothermal treatment, and had no significant side effects on the main tissues and organs. This strategy provided a new idea for the construction and clinical application of triple-combination therapeutic system as effective treatment for drug resistant triple negative breast cancer.

Abstract P4-01-06: LP-184, an acylfulvene class small molecule therapeutic, is synthetically lethal in HR deficient and PARP inhibitor resistant triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer accounting for approximately 15% to 20% of all newly diagnosed breast cancer cases. TNBCs are defined by lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2).Due to its special molecular phenotype, it is not sensitive to endocrine therapy or targeted therapy. Therefore, chemotherapy such as anthracyclines or taxanes is the main systemic treatment, but the efficacy of conventional postoperative adjuvant chemoradiotherapy is poor. ~35% of TNBC tumors show abnormalities in the HR pathway, making them sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) and DNA-damaging agents. Although more than 40% BRCA1/2-deficient patients fail to respond to PARPi and a substantial proportion of patients acquire PARPi resistance over time. TNBCs heterogeneous nature, poor prognosis and limited treatment options presents the urgent need of development of novel agents targeting tumor specific alterations. Lantern Pharma is advancing LP-184, an acylfulvene-derived prodrug that is specifically activated in tumors that overexpress the oxidoreductase enzyme Prostaglandin Reductase 1 (PTGR1), for the treatment of solid tumor indications including TNBC. LP-184 has multiple mechanisms of action (MOA). While It is synthetically lethal in tumors harboring DNA damage repair defects including Homologous Recombination (HR) deficiencies, it can also interrupt transcription. LP-184 efficacy was tested in a panel of breast cancer cell lines and patient derived TNBC xenografts models, both sensitive as well as resistant to PARP inhibitors and anthracyclines. LP-184 demonstrated nanomolar potency in six NCI-60 breast cancer cell lines (median IC50 = 327 nM). Subcutaneous patient-derived TNBC xenograft mouse models were used to determine tumor volume responses to LP-184 treatment in vivo. Xenograft tumors were derived from 10 treatment-naive HR deficient (HRD score > 50) primary TNBC patients with known BRCA1/2 loss of heterozygosity (LOH), 7 of which subsequently progressed on PARP inhibitor Olaparib. LP-184 (4mg/kg i.v., (q2d x 5 then 7 days off)x2) led to complete and durable regression in all 10 TNBC HRD-PDX models tested as compared to control (p < 0.0001). A tumor growth inhibition range of 107-141% was achieved across all the 10 models. For LP-184, T/C at the control group end day was 0% in 10/10 models, whereas across the same models for Olaparib, T/C was 0% in 2/10 models and ranged from 15 - 90% in 8/10 models. Terminal body weight change was only a transient weight loss < 4% with LP-184 treatment across all models. LP-184 exhibited superior potency than olaparib in TNBC PDX models that carry HRD mutations including PARP resistant models and was well tolerated in mice. As acylfulvene-induced damage is primarily repaired by transcription-coupled nucleotide-excision repair (TC-NER) and HR pathways, response to LP-184 is influenced by tumor DNA damage repair pathway status. Recent data highlight an important role of super enhancer driven core transcription regulatory circuits in the pathogenesis of TNBCs. Our results support the superior efficacy of LP-184 in TNBCs, likely linked to the multiple MOAs, and establish LP-184 as a promising new agent for future clinical testing in TNBC patients. We finally propose that LP-184 may be broadly efficacious in solid tumors with HR and/or TC-NER pathway defects, such as pancreatic, prostate, ovarian and bladder cancers. Citation Format: Aditya Kulkarni, Neha Biyani, Laura Brullé-Soumaré, Stefano Cairo, Panna Sharma, Kishor Bhatia. LP-184, an acylfulvene class small molecule therapeutic, is synthetically lethal in HR deficient and PARP inhibitor resistant triple negative breast cancer [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 P4-01-06.