Steen, Travis Vander

Abstract Background: Fatty Acid Synthase (FASN) is a key lipogenic enzyme co-regulated with HER2 and highly expressed in ER+ and HER2+ BC. Its targeting downregulates HER2 expression. Our preclinical studies revealed that FASN inhibition can sensitize or restore sensitivity to trastuzumab, paclitaxel and ET in HER2+ BC models. Denifanstat (deni, formerly TVB-2640) is a novel FASN inhibitor that demonstrated promising clinical activity and safety in a prior phase I trial as either monotherapy or in combination with paclitaxel. The primary objective of this study was to evaluate the objective tumor response rate (ORR) of deni plus trastuzumab (H) in combination with paclitaxel (T) or ET. Methods: Two independent, phase II clinical trials were initiated to assess the efficacy and safety of deni plus H in combination with T (Cohort A) or ET (Cohort B). Key inclusion criteria included HER2+ MBC resistant to HER2-directed therapy, measurable disease, unlimited ET, ≤6 prior chemotherapy or antibody drug conjugate regimens, and confirmation of HER2+ MBC on a pre-registration biopsy. Deni was administered 100 mg/m2 orally once daily. HP or H plus ET (fulvestrant or aromatase inhibitor) was administered at standard dose and schedule. Blood was collected on Cycle 1 Day 1 (C1D1), C1D8 and C2D1 for serum FASN analysis. An additional MBC tumor biopsy was collected on C2D1 for correlative studies. A 2-stage Simon design with a safety-run in was used to test the null hypothesis that the true ORR is at most 5% against the alternative it is at least 20%. With a sample size of 32 patients (pts) per cohort, if ≥4 responses are seen, this design has a 90% chance of rejecting the true ORR is <5% when the true ORR is ≥20% at a 0.10 level of significance. The decision to close both trials to enrollment early was multifactorial. Results: From August 2017 - February 2024, 33 pts with HER2+ MBC pre-registered, among which 17 were eligible and 16 were evaluable (15 pts in Cohort A and 1 pt in Cohort B). The most common reason for ineligibility was HER2-negative status on central review of the pre-registration biopsy. Median age was 57 years (range 35-80). All pts were non-Hispanic and 13 (81.3%) were White. Median prior lines of therapy was 5 (range 0 - 11). In Cohort A, there were 5 partial responses; thus, the primary endpoint was met with the ORR estimated to be 33.3% (90% CI: 14.2-57.7%). The median PFS time was 7.0 months (95 %CI: 3.7 - 11.3), and median OS was 25.7 months (95%CI: 10.2 - NE). No dose limiting toxicities were observed during the C1 safety run-in (6 pts). In Cohort A, 7 (46.7%) developed a grade 3 or 4 adverse event (AE) attributed to combination therapy. Grade ≥2 AEs regardless of attribution occurring in ≥20% of all pts included anemia, fatigue, neutropenia, palmar plantar erythrodysesthesia (PPE), and peripheral neuropathy. Treatment was discontinued due to progression (12; 80%) or AE (3; 20% - pneumonitis; neuropathy; PPE). In Cohort B, the pt maintained stable disease through 8 cycles with grade 3 ALT increase, and she remained alive 31.2 months post-registration. Median C1D1 FASN level was 32.7 ng/mL (IQR: 18.7 - 49.6 ng/mL). There was minimal association between C1D8 and C2D1 FASN levels and time to disease progression (C1D8 n=9, Spearman rank correlation coefficient (r s) = 0.040; C2D1 n=13, r s = -0.019). Analyses of changes in MBC tumor expression of FASN, phospho-AKT, and phospho-S6 are in progress. Conclusion: Deni combined with HP provided clinical antitumor activity in pts with HER2+ MBC resistant to trastuzumab. Expected paclitaxel-associated AEs were observed. Nearly half of pts experienced a grade 3 or 4 AE attributed to the combination therapy, and 20% stopped treatment due to AE. Further study of deni, H plus ET is necessary to draw safety and efficacy conclusions. Citation Format: T. C. Haddad, V. J. Suman, R. R. Roa, B. J. Ernst, R. Lupu, T. Vander Steen, M. H. Solanki, M. Keeney, D. W. Northfelt, K. S. Anderson, T. J. Hobday, S. Chumsri, J. E. Hoppenworth, J. L. Carroll, K. V. Giridhar, C. C. O’Sullivan, R. A. Leon-Ferre, M. P. Goetz. Phase II Trial of the FASN Inhibitor, Denifanstat (TVB-2640), Plus Trastuzumab in Combination with Paclitaxel or Endocrine Therapy (ET) in Patients with HER2+ Metastatic Breast Cancer (MBC) Resistant to Trastuzumab [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS5-01-16.

The initial excitement generated more than two decades ago by the discovery of drugs targeting fatty acid synthase (FASN)‐catalyzed de novo lipogenesis for cancer therapy was short‐lived. However, the advent of the first clinical‐grade FASN inhibitor (TVB‐2640; denifanstat), which is currently being studied in various phase II trials, and the exciting advances in understanding the FASN signalome are fueling a renewed interest in FASN‐targeted strategies for the treatment and prevention of cancer. Here, we provide a detailed overview of how FASN can drive phenotypic plasticity and cell fate decisions, mitochondrial regulation of cell death, immune escape and organ‐specific metastatic potential. We then present a variety of FASN‐targeted therapeutic approaches that address the major challenges facing FASN therapy. These include limitations of current FASN inhibitors and the lack of precision tools to maximize the therapeutic potential of FASN inhibitors in the clinic. Rethinking the role of FASN as a signal transducer in cancer pathogenesis may provide molecularly driven strategies to optimize FASN as a long‐awaited target for cancer therapeutics.

The identification of clinically important molecular mechanisms driving endocrine resistance is a priority in estrogen receptor-positive (ER+) breast cancer. Although both genomic and non-genomic cross-talk between the ER and growth factor receptors such as human epidermal growth factor receptor 2 (HER2) has frequently been associated with both experimental and clinical endocrine therapy resistance, combined targeting of ER and HER2 has failed to improve overall survival in endocrine non-responsive disease. Herein, we questioned the role of fatty acid synthase (FASN), a lipogenic enzyme linked to HER2-driven breast cancer aggressiveness, in the development and maintenance of hormone-independent growth and resistance to anti-estrogens in ER/HER2-positive (ER+/HER2+) breast cancer. The stimulatory effects of estradiol on FASN gene promoter activity and protein expression were blunted by anti-estrogens in endocrine-responsive breast cancer cells. Conversely, an AKT/MAPK-related constitutive hyperactivation of FASN gene promoter activity was unaltered in response to estradiol in non-endocrine responsive ER+/HER2+ breast cancer cells, and could be further enhanced by tamoxifen. Pharmacological blockade with structurally and mechanistically unrelated FASN inhibitors fully impeded the strong stimulatory activity of tamoxifen on the soft-agar colony forming capacity—an in vitro metric of tumorigenicity—of ER+/HER2+ breast cancer cells. In vivo treatment with a FASN inhibitor completely prevented the agonistic tumor-promoting activity of tamoxifen and fully restored its estrogen antagonist properties against ER/HER2-positive xenograft tumors in mice. Functional cancer proteomic data from The Cancer Proteome Atlas (TCPA) revealed that the ER+/HER2+ subtype was the highest FASN protein expressor compared to basal-like, HER2-enriched, and ER+/HER2-negative breast cancer groups. FASN is a biological determinant of HER2-driven endocrine resistance in ER+ breast cancer. Next-generation, clinical-grade FASN inhibitors may be therapeutically relevant to countering resistance to tamoxifen in FASN-overexpressing ER+/HER2+ breast carcinomas.

Sustained HER2/HER3 signaling due to the overproduction of the HER3 ligand heregulin (HRG) is proposed as a key contributor to endocrine resistance in estrogen receptor-positive (ER+) breast cancer. The molecular mechanisms linking HER2 transactivation by HRG-bound HER3 to the acquisition of a hormone-independent phenotype in ER+ breast cancer is, however, largely unknown. Here, we explored the possibility that autocrine HRG signaling drives cytokine-related endocrine resistance in ER+ breast cancer cells. We used human cytokine antibody arrays to semi-quantitatively measure the expression level of 60 cytokines and growth factors in the extracellular milieu of MCF-7 cells engineered to overexpress full-length HRGβ2 (MCF-7/HRG cells). Interleukin-8 (IL-8), a chemokine closely linked to ER inaction, emerged as one the most differentially expressed cytokines. Cytokine profiling using structural deletion mutants lacking both the N-terminus and the cytoplasmic-transmembrane region of HRGβ2—which is not secreted and cannot transactivate HER2—or lacking a nuclear localization signal at the N-terminus—which cannot localize at the nucleus but is actively secreted and transactivates HER2—revealed that the HRG-driven activation of IL-8 expression in ER+ cells required HRG secretion and transactivation of HER2 but not HRG nuclear localization. The functional blockade of IL-8 with a specific antibody inversely regulated ERα-driven transcriptional activation in endocrine-sensitive MCF-7 cells and endocrine-resistant MCF-7/HRG cells. Overall, these findings suggest that IL-8 participates in the HRG-driven endocrine resistance program in ER+/HER2- breast cancer and might illuminate a potential clinical setting for IL8- or CXCR1/2-neutralizing antibodies.

HER2 transactivation by the HER3 ligand heregulin (HRG) promotes an endocrine-resistant phenotype in the estrogen receptor-positive (ER+) luminal-B subtype of breast cancer. The underlying biological mechanisms that link them are, however, incompletely understood. Here, we evaluated the putative role of the lipogenic enzyme fatty acid synthase (FASN) as a major cause of HRG-driven endocrine resistance in ER+/HER2-negative breast cancer cells. MCF-7 cells engineered to stably overexpress HRG (MCF-7/HRG), an in vitro model of tamoxifen/fulvestrant-resistant luminal B-like breast cancer, showed a pronounced up-regulation of FASN gene/FASN protein expression. Autocrine HRG up-regulated FASN expression via HER2 transactivation and downstream activation of PI-3K/AKT and MAPK-ERK1/2 signaling pathways. The HRG-driven FASN-overexpressing phenotype was fully prevented in MCF-7 cells expressing a structural deletion mutant of HRG that is sequestered in a cellular compartment and lacks the ability to promote endocrine-resistance in an autocrine manner. Pharmacological inhibition of FASN activity blocked the estradiol-independent and tamoxifen/fulvestrant-refractory ability of MCF-7/HRG cells to anchorage-independently grow in soft-agar. In vivo treatment with a FASN inhibitor restored the anti-tumor activity of tamoxifen and fulvestrant against fast-growing, hormone-resistant MCF-7/HRG xenograft tumors in mice. Overall, these findings implicate FASN as a key enabler for endocrine resistance in HRG+/HER2- breast cancer and highlight the therapeutic potential of FASN inhibitors for the treatment of endocrine therapy-resistant luminal-B breast cancer.

Abstract Pancreatic cancer is the fourth leading cause of cancer related deaths in the United States and continues to be hindered with limited treatment options. One of the current therapeutic regimens for pancreatic cancer often include the nucleoside analog gemcitabine (Gem), while it has been used for many years, the overall survival rate lingers around 6%. New therapies are needed to treat this disease. Fatty acid synthase (FASN) has received increased attention as a potential target for cancer therapy in the last decade. FASN is the key enzyme in the de novo synthesis of palmitate from malonyl-CoA. Palmitate is the precursor other long-chain fatty acids (FA). However, the precise mechanism by which pharmacological interference with endogenous FA biogenesis might facilitate apoptosis in tumor cells remains unresolved. We herein explored the molecular relationship between FASN activity, oxidative stress/redox balance, and the intrinsic apoptotic pathway in FASN overexpressing-dependent pancreatic cancer cells. About 68% of Pancreatic Ductal Carcinomas (PDA) expresses high levels of Fatty acid synthase (FASN), and its expression is correlated with disease free survival. A newly developed FASN inhibitor with excellent pharmaceutical properties was used for our studies TVB-3166 (in vitro) and TVB-3664 (in vivo) and ten different PDX derived cells were used for the studies. Pharmacological blockade of FASN activity significantly increased the NADPH/NADP+ ratio, promoted the production of reactive oxygen species (ROS). Accordingly, the inhibition of endogenous FA synthesis promoted mitochondrial membrane permeabilization, cytochrome c release, and apoptotic cell death. Each step of the FASN inhibition/BH3-only protein axis could be reversed with a ROS scavenger or the FASN end-product palmitate, thus confirming that the delineated pro-apoptotic cascade likely reflects on-target effects of FASN inhibition. Most importantly, the in vivo studies using PaCa-PDX reveal a significant decrease in tumor growth and furthermore a synergistic effect of between TVB-3664 and Gem. In conclusion, our findings uncover a novel FASN-dependent mitochondrial priming that links de novo FA biosynthesis with the intrinsic apoptotic threshold in cancer cells. The discovery that FASN-inhibited cancer cells exist in an apoptosis-prone state highly sensitive to BH3 mimetics warrants clinical exploration in FASN-overexpressing pancreatic cancer patients. Citation Format: Travis Vander Steen, Li Ding, Yu Zhang, George Kemble, Daniel Billadeau, Ruth Lupu. Fatty acid synthase: A new therapeutic target for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3723.

Abstract Fatty Acid Synthase (FASN), the key multienzyme in de novo lipogenesis, is upregulated and activated in numerous carcinomas, including breast cancer. Fatty acids play a fundamental role in cellular structure, energy production and storage and are intermediates in the biosynthesis of hormones and other biologic molecules. Therefore, FASN is a novel therapeutic target for treatment in cancer cells that have upregulated FASN protein. BT474 breast cancer cells, which overexpress FASN, were treated with a pharmacologic FASN inhibitor and resulted in apoptosis in a dose-dependent manner. Inhibition of FASN simultaneously upregulates the pro-apoptotic BCL2 family members Bim, Puma, and Noxa; additionally, FASN inhibition alters the NDAPH/NADP+ balance and induces reactive oxygen species (ROS) production. Most significantly, FASN inhibition induces sensitization of breast cancer cells to the BH3 mimetic navitoclax, a potential opportunity to use BH3 mimetics for the treatment of breast carcinomas. These results support further preclinical and potentially clinical development of a dual FASN and Bcl-2 treatment for breast cancer tumors that express FASN. In addition to apoptosis, there is an increase in cellular senescence in breast cancer cells overexpressing FASN when treated with FASN inhibitors. The increase in ROS, a known effect of FASN inhibition, is known to induce cellular stress, resulting in both apoptosis and senescence. Increased senescence was initially observed by x-gal staining of BT474 cells treated with FASN inhibitors via microscopy and FLOW. We also observed an increased expression of several pro-senescent genes both at the protein and mRNA levels, including p16/INK4a, p14ARF/p19ARF, p15, and p21. A hallmark of age-induced cellular senescence is the shortening of telomeres. However, telomere shortening was not observed, suggesting that senescence was a result of FASN inhibition and activation of senescence-induced genes. Therefore, inhibition of FASN appears to be an ideal target for therapeutic treatment of breast cancers that overexpress FASN through both apoptosis and senescence. Citation Format: Travis Vander Steen, Ruth Lupu. Inhibition of FASN induces apoptosis and senescence in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 318.