Preclinical Triple-negative Breast Cancer Research Articles

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.

Loss of tumor suppressors promotes inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression

Low response rate, treatment relapse, and resistance remain key challenges for cancer treatment with immune checkpoint blockade (ICB). Here we report that loss of specific tumor suppressors (TS) induces an inflammatory response and promotes an immune suppressive tumor microenvironment. Importantly, low expression of these TSs is associated with a higher expression of immune checkpoint inhibitory mediators. Here we identify, by using in vivo CRISPR/Cas9 based loss-of-function screening, that NF1, TSC1, and TGF-β RII as TSs regulating immune composition. Loss of each of these three TSs leads to alterations in chromatin accessibility and enhances IL6-JAK3-STAT3/6 inflammatory pathways. This results in an immune suppressive landscape, characterized by increased numbers of LAG3+ CD8 and CD4 T cells. ICB targeting LAG3 and PD-L1 simultaneously inhibits metastatic progression in preclinical triple negative breast cancer (TNBC) mouse models of NF1-, TSC1- or TGF-β RII- deficient tumors. Our study thus reveals a role of TSs in regulating metastasis via non-cell-autonomous modulation of the immune compartment and provides proof-of-principle for ICB targeting LAG3 for patients with NF1-, TSC1- or TGF-β RII-inactivated cancers.

Ultrasound Controllable Release of Proteolysis Targeting Chimeras for Triple-Negative Breast Cancer Treatment.

Triple-negative breast cancer (TNBC) is a special subtype of breast cancer, which is highly aggressive and incurable. Here, we proposed an ultrasound activatable bromodomain-containing protein 4 (BRD4) proteolysis targeting chimera (PROTAC) release strategy for the first time for precisely controlled protein degradation in preclinical TNBC model. Through combination of PROTAC and ultrasound-targeted microbubble destruction (UTMD) technology, the present strategy also aims to concurrently solve the major limitations of poor loading capacity of microbubbles and undesirable targeting and membrane permeability of PROTAC. PROTAC (ARV-825)-encapsulated microbubbles, ARV-MBs, were developed for the efficacious treatment of TNBC invitro and invivo. The microbubbles we synthesized showed ultrasound-responsive drug release ability, which could effectively promote the penetration of PROTAC into tumor site and tumor cell. Under ultrasound, ARV-MBs could play an effective antitumor effect by potentiating the ubiquitination and degradation of BRD4 in tumor. The current study may provide a new idea for promoting clinical translation of drug-loaded microbubbles and PROTAC, and offer a new efficacious therapeutic modality for TNBC.

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.

Abstract B007: Unexpected synthetic lethality mechanisms in eIF4A-targeted therapy

Abstract BACKGROUND Dysregulation of mRNA translation, which is prevalent in cancer, presents an underexplored therapeutic vulnerability. The eukaryotic translation initiation factor 4A (eIF4A) promotes protein synthesis by unwinding secondary structures in the 5’-untranslated region of mRNAs to facilitate translation initiation. While perturbation of eIF4A minimally affects the health normal cells, it is crucial for the translation of many oncogenes and is considered essential in most cancer cell lines. Zotatifin, a first-in-class eIF4A inhibitor, is currently under evaluation in a Phase I/II clinical trial for KRAS-mutant tumors and ER+ breast cancers. In addition to the tumor-intrinsic effect, our recent publication in the Journal of Clinical Investigation revealed that pharmacological targeting of eIF4A not only affects tumor cells but also improves the tumor immune microenvironment. This results in tumor inhibition, heightened sensitivity to immune checkpoint blockade, and remarkable synergism with platinum in various preclinical triple-negative breast cancer mouse models. OBJECTIVES To investigate the mechanism underlying the synergy between eIF4A-targeted therapy and platinum. UNPUBLISHED RESULT Mass spectrometry proteomic analysis coupled with ELISA and qPCR analysis unveiled that eIF4A-targeted therapy promotes type I interferon (IFN) secretion and induces a robust type I IFN response both in vivo and in vitro. This suggests a tumor cell-autonomous effect of IFN induction by eIF4A inhibition. Combining platinum with eIF4A-targeted therapy further heightens the type I IFN response in vivo, leading to T cell-and macrophage-dependent synergistic tumor regression. Interestingly, this synergy was not observed in vitro, indicating the involvement of the host immune system in the efficacy of combination therapy. It is possible that platinum may stimulate certain immune populations to secrete IFN, which acts on tumor cells to upregulate their IFN response. CONCLUSION Activation of the type I IFN response by eIF4A inhibition combined with platinum in vivo can lead to synthetic lethality of tumor cells. These studies not only offer novel insights into eIF4A biology but also provide valuable guidance for ongoing and future clinical trials. Citation Format: Na Zhao, Elena Kabotyanski, Alexander Saltzman, Jeffrey Rosen. Unexpected synthetic lethality mechanisms in eIF4A-targeted therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B007.

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.

FXR Agonism with Bile Acid Mimetic Reduces Pre-Clinical Triple-Negative Breast Cancer Burden.

Bariatric surgery is associated with improved outcomes for several cancers, including breast cancer (BC), although the mechanisms mediating this protection are unknown. We hypothesized that elevated bile acid pools detected after bariatric surgery may be factors that contribute to improved BC outcomes. Patients with greater expression of the bile acid receptor FXR displayed improved survival in specific aggressive BC subtypes. FXR is a nuclear hormone receptor activated by primary bile acids. Therefore, we posited that activating FXR using an established FDA-approved agonist would induce anticancer effects. Using in vivo and in vitro approaches, we determined the anti-tumor potential of bile acid receptor agonism. Indeed, FXR agonism by the bile acid mimetic known commercially as Ocaliva ("OCA"), or Obeticholic acid (INT-747), significantly reduced BC progression and overall tumor burden in a pre-clinical model. The transcriptomic analysis of tumors in mice subjected to OCA treatment revealed differential gene expression patterns compared to vehicle controls. Notably, there was a significant down-regulation of the oncogenic transcription factor MAX (MYC-associated factor X), which interacts with the oncogene MYC. Gene set enrichment analysis (GSEA) further demonstrated a statistically significant downregulation of the Hallmark MYC-related gene set (MYC Target V1) following OCA treatment. In human and murine BC analyses in vitro, agonism of FXR significantly and dose-dependently inhibited proliferation, migration, and viability. In contrast, the synthetic agonism of another common bile acid receptor, the G protein-coupled bile acid receptor TGR5 (GPBAR1) which is mainly activated by secondary bile acids, failed to significantly alter cancer cell dynamics. In conclusion, agonism of FXR by primary bile acid memetic OCA yields potent anti-tumor effects potentially through inhibition of proliferation and migration and reduced cell viability. These findings suggest that FXR is a tumor suppressor gene with a high potential for use in personalized therapeutic strategies for individuals with BC.

Abstract 1187: Sensitization to chemo-immunotherapy by targeting TGF-β in preclinical triple negative breast cancer models

Abstract Metastatic triple-negative breast cancer (mTNBC) is refractory to anti-PD-(L)1 immunotherapy. In order to sensitize it, preclinical data show the value of adding chemotherapy treatment for its ability to induce immunogenic cancer cell death (Kroemer et al, 2013). This would enable the recruitment of cytotoxic T-CD8+ lymphocytes (CTL) and thus initiate an antitumor immune response to sensitize tumors to immunotherapy. Nevertheless, the results of clinical trials using chemo-immunotherapy combinations are divergent (IMpassion 130 and 131). The IMpassion 130 trial demonstrated the efficacy of nab-paclitaxel and atezolizumab (anti-PD-L1) in PD-L1+ patients, but unfortunately, IMpassion 131 with paclitaxel did not replicate these results. The choice of chemotherapy to combine with immunotherapy appears to be crucial for inducing an antitumor immune response. Optimizing chemo-immunotherapy combinations is a major challenge in the management of TNBC. The initial aim of this project was to evaluate the immune effects of an experimental chemotherapy doublet, based on two drugs used in mTNBC: cisplatin (CDDP) and eribulin. The idea was to find an alternative to the use of taxanes in combination with anti-PD-(L)1.The effect of the two molecules, alone or in combination, was evaluated on immunogenic death stigmata, modulation of effector or immunosuppressive immune populations and stromal populations, and sensitization to anti-PD-L1 immunotherapy in the immunologically "cold" model: 4T1. The in vivo studies has been realized by generation of orthotopic 4T1 tumors, the analysis of the immune infiltration was performed by RT-qPCR, IHC and flow cytometry. In vitro, our results indicate that CDDP, and more intensely in combination with eribulin, significantly induces the various immunogenic death stigmas. In vivo, the therapeutic effect of chemotherapies is limited, with a superior effect of CDDP and the combination. Nevertheless, CTL recruitment and functionality induced by CDDP are significantly enhanced by eribulin. Despite these data pointing to a potential therapeutic synergy with anti PD-L1 immunotherapy, chemo-immunotherapy treatment fails to induce a superior treatment effect. The significant increase in a TGF-β signature induced by the combination in parallel with CTL recruitment explains this resistance. The results show that inhibition of TGF-β, combined with chemo-immunotherapy in the 4T1 model, leads to a reduction in tumor immunosuppression and intratumoral fibrosis, as well as an increase in CTL infiltration and cytotoxicity, enabling tumor sensitization. Overall, this project highlights the benefits of using an experimental doublet of CDDP and eribulin-based chemotherapies. This project demonstrates that management of chemo-induced immunosuppression mechanisms is necessary to overcome tumor resistance to immunotherapy. Citation Format: Laura Kalfeist, Fanny Ledys, Stacy Petit, Samia Kada-Mohammed, Mickael Rialland, François Ghiringhelli, Emeric Limagne, Sylvain Ladoire. Sensitization to chemo-immunotherapy by targeting TGF-β in preclinical triple negative breast cancer models [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 1187.

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 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.

SYST-13 PHASE II STUDY OF THE COMBINATION OF LIPOSOMAL IRINOTECAN AND PEMBROLIZUMAB FOR TRIPLE-NEGATIVE BREAST CANCER (TNBC) WITH BRAIN METASTASES (BM)

Abstract BACKGROUND Breast cancer is one of the most common cancers associated with brain metastases (BM). Up to 50% of patients with metastatic triple-negative breast cancer (TNBC) develop BM which portends a poor prognosis, with a median survival of 4.4-7.3 months. There is a lack of effective systemic therapy. Irinotecan is a topoisomerase-1 inhibitor with a response rate of 5-23% in advanced breast cancer. Nal-IRI is an intravenous liposomal formulation of irinotecan, with greater efficacy in tumor growth inhibition and the ability to cross the blood brain barrier (BBB) than irinotecan, resulting in a prolonged survival in preclinical TNBC BM models. Additionally, Nal-IRI has demonstrated promising anti-tumor activity in patients with TNBC- BM in a phase-I trial (NCT01770353). Pembrolizumab is a humanized anti-PD-1 monoclonal antibody which has shown efficacy in TNBC. The purpose of this study is to evaluate whether the combination of Nal-IRI and pembrolizumab can provide a synergic effect to control the CNS disease and prolong survival in TNBC with progressive BM. METHODS This is a phase II, single arm trial with a safety lead-in to evaluate the efficacy of nal-IRI (50-70mg/m2 IV Q2W) in combination with pembrolizumab (400mg IV Q6W). Simon’s 2-stage design is used, with 18 patients in the 1st stage and additional 24 at 2nd stage for a total of 42. The first 6 patients will serve as a safety lead-in. Key eligibilities include: histologically/cytologically confirmed TNBC with new or progressive BM; prior immunotherapy is allowed but not prior nal-IRI/irinotecan; prior sacituzumab-govitecan is permitted if disease stable for ≥16-week while on therapy and ≥24-week washout prior to starting trial; measurable disease; and ≤4 prior lines of therapy in the metastatic setting. The primary endpoint is CNS disease control rate (DCR) at 6 months using RANO-BM criteria. Secondary endpoints include CNS and non-CNS ORR, PFS and OS. (ClinicalTrials.gov: NCT05255666)

Abstract CT131: RVU120 SOL-021: An open-label, single agent, phase I/II trial of RVU120 (SEL120) in patients with relapsed/refractory metastatic or advanced solid tumors

Abstract Background: CDK8 and its paralog CDK19 have central roles in maintenance of cancer cell viability and undifferentiated state for a variety of tumor types. (Dannappel et al. 2019; Rzymski et al. 2015; Philip et al. 2018). CDK8 and CDK19 work by incorporating in a mutually exclusive manner, into a Mediator kinase module, that reversibly associates with a protein complex regulator of RNA Polymerase II, to mediate gene transcription. Targeting cancer-specific gene transcription via CDK8/19 inhibition has potential for treatment of solid tumors. RVU120 (SEL120) is a selective CDK8/19 inhibitor with preclinical efficacy in hematologic malignancies and a variety of solid tumor types (Rzymski et al. 2017). RVU120 showed strong anticancer activity in preclinical triple-negative breast cancer (TNBC) models, especially in the mesenchymal stem-like (MSL) molecular subtype (Rzymski et al SABCS 2021). Methods: SOL-021 is a Phase I/II clinical trial of RVU120 in Patients with metastatic or advanced solid tumors, who have exhausted available treatments, currently ongoing in Poland and Spain under NCT05052255 clinical trial register number. RVU120 is administered as single oral dose every other day (QOD) for a total of 7 doses on Days 1, 3, 5, 7, 9, 11 and 13, in a 3-week treatment cycle until progression or unacceptable toxicity. The study is designed in 2 parts: Part 1 follows a 3+3 dose escalation design and has primary objectives to characterize the safety and tolerability of RVU120 (SEL120) as single agent in patients with different tumors types and determination of the RP2D. Secondary objectives include objective response rate (ORR) according to RECIST criteria v1.1, duration of response (DoR), disease-control rate, progression-free survival (PFS), overall survival (OS) and pharmacokinetic profile of RVU120.Part 2 will primarily explore the anti-tumor activity of RVU120 as single agent in different patient populations. Based on strong preclinical evidence of anticancer activity of RVU120 on TNBC cells (Tomasz Rzymski SABCS #1766), patients with relapsed/refractory TNBC will be enrolled. Additionally, relapsed/refractory patients agnostic of their solid tumor type and selected based on their biomarker profile will be enrolled. The total duration of the study is approximately 27 months (12 and 15 for part 1 and 2, respectively). Results: At 11 Feb 2022 data cut off, 5 patients have been enrolled in part 1 of the study and no SAE have been observed: First cohort, 75 mg, was closed on 24 Nov 2021 with no DLT observed; current ongoing cohort, 100 mg dose level, is expected to be closed in the upcoming week, and subsequent cohort, 125 mg, to be opened mid-March. Conclusion: The trial is continuing as planned; the next data analysis will occur first week of March 2022. Citation Format: Rafal Dziadziuszko, Elena Garralda, Noemi Angelosanto, Tomasz Rzymski, Renata Dudziak, Krzysztof Brzózka, Peter Littlewood, Hendrik Nogai, Axel Glasmacher, Iwona Lugowska. RVU120 SOL-021: An open-label, single agent, phase I/II trial of RVU120 (SEL120) in patients with relapsed/refractory metastatic or advanced solid tumors [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 CT131.

Phase I/II trial of RVU120 (SEL120), a CDK8/CDK19 inhibitor in patients with relapsed/refractory metastatic or advanced solid tumors.

e15091 Background: CDK 8 /CDK19 are kinases involved in transcriptional control of embryonic development and cellular homeostasis via the mediator complex. They are also described to maintain tumor dedifferentiation and stem cell properties. A variety of cancer cells hijack the mediator complex and targeting cancer-specific gene transcription via CDK8/19 inhibition has potential for treatment of solid tumors. RVU120 (SEL120) is a selective CDK8/19 inhibitor with preclinical efficacy in hematologic malignancies and a variety of solid tumor types (Rzymski et al. 2017). RVU120 showed strong anticancer activity in preclinical triple-negative breast cancer (TNBC) models, especially in the mesenchymal stem-like (MSL) molecular subtype (Rzymski et al SABCS 2021). Methods: A Phase I/II clinical trial of RVU120 in solid tumors is currently ongoing (NCT05052255). Patients with metastatic or advanced solid tumors without available treatments receive increasing doses of RVU120 as a single oral dose every other day (QOD) for a total of 7 doses on Days 1, 3, 5, 7, 9, 11 and 13, in a 3-week treatment cycle. Primary objectives of the study are safety / tolerability of RVU120 as single agent and determination of the recommended Phase II dose (RP2D) via a 3+3 dose escalation design. Secondary objectives include objective response rate (ORR) (RECIST criteria v1.1) and PK of RVU120. Results: As of 11FEB 2022, 5 pts have been enrolled into the trial: 3 into cohort 1, 75 mg dose, 2 into cohort 2, 100 mg. None of the pts experienced a DLT, SAEs or > Grade 3 AE. Treatment-related AEs were all G1 except for G2 diarrhea and weakness. Cohort 1 patients had at least 1 post-baseline tumor assessment. A 62 YO patient, stage IV esophago-gastric junction carcinoma, metastases to the liver, retroperitoneum, adrenal gland and portocaval lymph nodes, progressing after 4 lines of previous therapy, achieved a SD with a +4% change of target lesion size and progressed in cycle 6. A 65 YO patient, stage IV adenoid cystic carcinoma of the trachea, metastases to the lung and refractory to antiandrogens and radiotherapy, achieved a SD with -4% change of target lesion size after 3 cycles and is currently ongoing in cycle 5. A 43 YO pt stage IV thymic carcinoma, metastases to the lung, mediastinum, pleura and lymph nodes, progressing under cisplatin/etoposide, stopped study drug at the end of cycle 3 for PD in non-target lesions, target lesions increased by 11%. Cohort 2 patients, a 65 YO pt with pancreatic cancer stage IV, metastases to liver and lung, and a 55 YO pt with TNBC stage IV, metastases to the lung, will be evaluated end of cycle 3. Conclusions: In the first dose escalation level with single agent RVU120, no DLTs or ≥G3 AEs were observed in patients with previously progressive solid tumors. Initial assessments in 3 pts demonstrated stable disease in 2 patients. Collection of further data at higher doses is ongoing with 2 patients enrolled at 100 mg. Clinical trial information: NCT05052255.

UBR5 promotes tumor immune evasion through enhancing IFN-γ-induced PDL1 transcription in triple negative breast cancer.

Background: The up-regulation of PD-L1 is recognized as an adaption of cancer cells to evade immune surveillance and attack. However, the intrinsic mechanisms of the induction of PD-L1 by interferon-γ (IFN-γ) in tumor microenvironment remain incompletely characterized. Ubiquitin ligase E3 component N-recognition protein 5 (UBR5) has a critical role in tumorigenesis of triple negative breast cancer (TNBC) by triggering specific immune responses to the tumor. Dual targeting of UBR5 and PD-L1 exhibited superior therapeutic benefits in a preclinical TNBC model in short term.Methods: The regulation of UBR5 to PD-L1 upon IFN-γ stimulation was evaluated through in UBR5 deficiency, reconstitution or overexpression cell line models by quantitative PCR, immunohistochemistry and RNA-seq. The effects of PD-L1 regulation by UBR5 and double blockade of both genes were evaluated in mouse TNBC model. Luciferase reporter assay, chromatin immunoprecipitation-qPCR and bioinformatics analysis were performed to explore the transcription factors involved in the regulation of UBR5 to PD-L1.Results: E3 ubiquitin ligase UBR5 plays a key role in IFN-γ-induced PDL1 transcription in TNBC in an E3 ubiquitination activity-independent manner. RNA-seq-based transcriptomic analyses reveal that UBR5 globally affects the genes in the IFN-γ-induced signaling pathway. Through its poly adenylate binding (PABC) domain, UBR5 enhances the transactivation of PDL1 by upregulating protein kinase RNA-activated (PKR), and PKR's downstream factors including signal transducers and activators of transcription 1 (STAT1) and interferon regulatory factor 1 (IRF1). Restoration of PD-L1 expression in UBR5-deficient tumor cells recoups their malignancy in vivo, whereas CRISPR/Cas9-mediated simultaneous abrogation of UBR5 and PD-L1 expression yields synergistic therapeutic benefits than either blockade alone, with a strong impact on the tumor microenvironment.Conclusions: This study identifies a novel regulator of PDL1 transcription, elucidates the underlying molecular mechanisms and provides a strong rationale for combination cancer immunotherapies targeting UBR5 and PD-L1.

Tumor suppressor PLK2 may serve as a biomarker in triple-negative breast cancer for improved response to PLK1 therapeutics.

Polo-like kinase (PLK) family members play important roles in cell cycle regulation. The founding member PLK1 is oncogenic and preclinically validated as a cancer therapeutic target. Paradoxically, frequent loss of chromosome 5q11-35 which includes PLK2 is observed in basal-like breast cancer. In this study, we found that PLK2 was tumor suppressive in breast cancer, preferentially in basal-like and triple-negative breast cancer (TNBC) subtypes. Knockdown of PLK1 rescued phenotypes induced by PLK2-loss both in vitro and in vivo. We also demonstrated that PLK2 directly interacted with PLK1 at prometaphase through the kinase but not the polo-box domains of PLK2, suggesting PLK2 functioned at least partially through the interaction with PLK1. Furthermore, an improved treatment response was seen in both Plk2-deleted/low mouse preclinical and PDX TNBC models using the PLK1 inhibitor volasertib alone or in combination with carboplatin. Re-expression of PLK2 in an inducible PLK2-null mouse model reduced the therapeutic efficacy of volasertib. In summary, this study delineates the effects of chromosome 5q loss in TNBC that includes PLK2, the relationship between PLK2 and PLK1, and how this may render PLK2-deleted/low tumors more sensitive to PLK1 inhibition in combination with chemotherapy.

Comparative Gene Signature of (-)-Oleocanthal Formulation Treatments in Heterogeneous Triple Negative Breast Tumor Models: Oncological Therapeutic Target Insights.

Triple negative breast cancer (TNBC) heterogeneity and limited therapeutic options confer its phenotypic aggressiveness. The discovery of anti-TNBC natural products with valid molecular target(s) and defined pharmacodynamic profile would facilitate their therapeutic nutraceutical use by TNBC patients. The extra-virgin olive oil (EVOO) is a key Mediterranean diet ingredient. S-(−)-Oleocanthal (OC) leads the bioactive anti-tumor EVOO phenolic ingredients. A previous study reported the solid dispersion formulated OC with (+)-xylitol (OC-X) suppressed the in vivo progression and recurrence of the TNBC MDA-MB-231 cells. This study investigates the ability of OC-X formulation to suppress the in vivo heterogeneous BC initiation and progression utilizing advanced preclinical transgenic MMTV-PyVT and TNBC PDX mouse models. Furthermore, the clustering of the gene expression profiles in MMTV-PyVT and PDX mouse tumors treated with OC-X acquired by a Clariom S microarray analysis identified the distinctly affected genes. Several affected novel signature genes identified in response to OC-X treatments and proved overlapped in both mouse and human tumor models, shedding some lights toward understanding the OC anticancer molecular mechanism and assisting in predicting prospective clinical outcomes. This study provides molecular and preclinical evidences of OC-X potential as a nutraceutical suppressing heterogeneous TNBC model and offers preliminary gene-level therapeutic mechanistic insights.

Abstract IA023: TIME and Age: Impact of age on the tumor immune microenvironment and response to therapy

Abstract Breast cancer affects women of all ages yet young (<40) and older women (>65) experience worse survival than their middle-aged counterparts. Age is associated with increasing immune dysfunction, yet the clinical implications of age-related changes are unknown, as women <40 and >65 years of age are under-represented in clinical trials. We hypothesized that both young and older immunological age impact breast cancer outcomes and present unique obstacles to therapeutic efficacy. Using pre-clinical triple-negative breast cancer (TNBC) models, we had found that bone marrow progenitor cells upregulate granulin (GRN), a secreted pro-inflammatory factor. Once mobilized from the marrow, those cells infiltrate tumors where GRN generates a tumor-supportive microenvironment. High GRN also correlated with reduced survival in our cohort of breast cancer patients. When we performed the same experiments in immunologically old mice (12-18 mos), tumors grew slower and GRN+ cell recruitment was reduced. GRN+ bone marrow cells from young donor mice rescued tumor growth in aged recipients, indicating that pro-tumor immunity resides in the marrow and explaining the aggressive TNBC growth in young cohorts. We next asked if age impacts immune checkpoint blockade (ICB) efficacy, using a 4T1 TNBC model to evaluate anti-PD-L1 or anti-CTLA4 monotherapy. Young mice responded to anti-PD-L1 and anti-CTLA4 with significantly prolonged survival; however, aged mice derived no benefit from ICB therapy. In response to ICB, tumors in young mice had significantly higher CD8+:Treg ratios and were enriched for inflammatory and interferon (IFN)-gamma response genes, thus revealing active anti-tumor immunity in young but not aged mice. Importantly, IFN-alpha, IFN-gamma, and inflammatory pathways were enriched in young relative to aged isotype control mice. When we analyzed METABRIC gene expression data for TNBC patients <40 and >65, IFNg, IFNa, and inflammatory responses were enriched only in patients <40. Hence, immune priming and T-cell inflamed phenotypes appear to be functional prior to treatment in young mice and are likewise enriched in women <40, but not >65. To stimulate immune priming in aged mice, we evaluated the STING agonist, DMXAA, which significantly prolonged survival in combination with ICB in the aged cohort. We are currently performing a pre-clinical study mirroring the IMpassion031 trial, which recently reported improved pathological complete response rates to neoadjuvant atezolizumab (anti-PD-L1)+nab-paclitaxel in TNBC. In a Met1 TNBC model, tumors grew slower with improved survival in the untreated aged mice. Young and aged mice responded to paclitaxel alone and anti-PD-L1 monotherapy; however, anti-PD-L1 provided additional benefit to paclitaxel only in young mice, similar to the trend reported in the IMpassion031 trial. Our results support a model whereby the net effects of pro- and anti-tumor immunity change with age and affect breast cancer outcomes. Understanding age-related immune function should enable us to design age-stratified breast cancer therapies. Citation Format: Sandra S. McAllister. TIME and Age: Impact of age on the tumor immune microenvironment and response to therapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA023.

Caloric Restriction Impairs Regulatory T cells Within the Tumor Microenvironment After Radiation and Primes Effector T cells.

Outcomes for triple negative breast cancer (TNBC) are poor and may be improved by increasing CD8+ tumor infiltrating lymphocytes (TIL) to augment antitumor immunity. Radiation (RT) can promote immunogenic cell death with increased antitumor T cell activity but also stimulates suppressive regulatory T cells (Tregs). Because metabolic alterations affect immune homeostasis and prior studies show caloric restriction (CR) combined with RT improves preclinical TNBC outcomes, we hypothesized that CR augments RT, in part, by altering intratumoral immunity. Using an invivo model of TNBC, we treated mice with ad libitum (AL) diet, radiation, a CR diet, or CR + RT, and demonstrated an immune suppressive environment with a significant increase in CD4+ CD25+Foxp3+ Tregs after RT but not in CR-fed mice. CD8:Treg ratio in CR + RT TIL increased 4-fold compared with AL + RT mice. Invivo CD8 depletion was performed to assess the role of effector T cells in mitigating the effects of CR, and it was found that in mice undergoing CR, depletion of CD8 T cells resulted in increased tumor progression and decreased median survival compared with isotype control-treated mice. In addition, PD-1 expression on CD3+CD8+ Tcells within the tumor microenvironment was significantly increased in CR + RT versus AL + RT treated mice as per immunofluorescence. Serum from breast cancer patients undergoing RT alone or CR and RT was collected pre- and postintervention, and a cytokine array demonstrated that patients treated with CR + RT had notable decreases in immunosuppressive cytokines such as IL-2Rγ, IL-10Rβ, and TGF-β2 and 3 compared with patients receiving RT alone. In conclusion, combining CR with RT decreases intratumoral Tregs, increases CD8:Treg, and increases PD-1 expression via a process dependent on CD8 T cells in a TNBC model. Breast cancer patients undergoing CR concurrently with RT also had significant reduction in immunosuppressive cytokine levels compared with those receiving RT alone.

Abstract SP090: Tumor INF signaling - A double edged sword in cancer

Abstract Our pre-clinical research using breast cancer models has revealed how certain inflammatory pathways can have opposing effects on primary disease and metastasis. We also learned that immunological age can impact response to therapy, leading to age-stratified outcomes. In one study, we developed pre-clinical models of triple-negative breast cancer (TNBC) lung metastasis. We uncovered mechanisms by which certain primary breast cancers can prevent their disseminated tumor cells from generating tumors. These primary tumors elicit a systemic innate immune response involving IL-1β-expressing neutrophils that infiltrate the lungs. Our group and others had previously established that IL-1β promotes primary breast cancer growth. However, we found that in the lungs, IL-1β maintains disseminated tumor cells in an undifferentiated, non-proliferative state. Inhibiting either inflammation or IL-1R signaling enables robust outgrowth of lung metastases. Importantly, we found that among patients with lymph node-positive breast cancer (i.e., those at high risk for metastasis) high IL-1β associates with improved overall survival and distant metastasis-free survival. Our findings reveal a double-edged sword in which IL-1R inhibitors might reduce primary disease but promote metastatic disease. To avoid this dichotomy, we are exploring strategies that prevent disseminated tumor cell differentiation, as was achieved here by IL-1β, to prevent metastatic outgrowth and provide a window of opportunity to target early metastases. As another example, we evaluated immune checkpoint blockade (ICB) therapy using various pre-clinical TNBC models in young and aged mice. We observed reduced tumor growth and improved survival in response to anti-PD-1 and anti-CTLA-4 treatment in young mice compared to aged mice. Immune profiling of peripheral blood and tumor samples demonstrated differences between young and aged mice treated with immune checkpoint blockade. To gain insights into the mechanisms behind those results, we performed whole tumor RNAseq on treated young versus aged mice. Notably, the most significantly enriched gene signatures included genes in interferon response pathways - with impaired responses in tumors of aged mice. Informed by those findings, we interrogated the METABRIC tumor gene expression database, stratifying TNBC patients < 40 and > 65 years of age. Two distinct clusters were identified, including enrichment of interferon-related genes in the "young" cohort. Turning back to our pre-clinical models, we used a drug that stimulates interferon response genes (STING agonists) in combination with ICB and found significantly improved survival in the aged cohorts. Our study demonstrated that age determines the T cell inflamed phenotype and affects response to immune checkpoint blockade in TNBC. Evaluating interferon-related genes from tumor genomic data may aid identification of patients for whom combination therapy including an interferon pathway activator with ICB may be required. These findings highlight the need to understand how inflammatory signatures should be interpreted in order to design more effective clinical phase trials and how pre-clinical models need to better mimic intent-to-treat populations. Citation Format: SS McAllister. Tumor INF signaling - A double edged sword in cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr SP090.

EGFR signaling promotes resistance to CHK1 inhibitor prexasertib in triple negative breast cancer.

Aim: Innate resistance to the CHK1 inhibitor prexasertib has been described, but resistance mechanisms are not understood. We aimed to determine the role epidermal growth factor receptor (EGFR) plays in innate resistance to prexasertib in triple negative breast cancer (TNBC).Methods: Using a panel of pre-clinical TNBC cell lines, we measured the sensitivity to prexasertib. We examined the effect activation of EGFR had on prexasertib sensitivity. We measured the synergy of dual blockade of EGFR with erlotinib and CHK1 with prexasertib in TNBC cell lines and xenografts.Results: EGFR overexpression and activation increased resistance to CHK1 inhibition by prexasertib. EGFR promoted the phosphorylation of BCL2-associated agonist of cell death (BAD), inactivating its pro-apoptotic functions. Inhibition of EGFR reversed BAD phosphorylation, increasing sensitivity to prexasertib.Conclusion: The use of prexasertib as a monotherapy in TNBC has been limited due to modest clinical responses. We demonstrated that EGFR activation contributes to innate resistance to prexasertib in TNBC and potentially other cancers. EGFR expression status should be considered in clinical trials examining prexasertib’s use as a monotherapy or combination therapy.