Detry, Anna

Abstract Background: Endocrine therapies remain central to estrogen receptor alpha-positive (ERα+) breast cancer treatment, yet their predominantly cytostatic effects highlight the need for combinatorial approaches that promote tumor cell death. We therefore tested BH3 mimetics as single agents and in combination with (Z)-endoxifen (ENDX) across endocrine sensitive and endocrine resistant ERα+ breast cancer models. Methods: Six BH3 mimetics were tested in MCF7 and T47D cells and matched ENDX-resistant and fulvestrant-resistant derivatives. ENDX combinations were assessed using 2D proliferation and synergy assays, 3D RASTRUM® bioprinted cultures, migration assays, and in vivo MCF7 xenograft studies. Results: BH3 mimetics showed broad anti-proliferative activity as single agents in endocrine sensitive and resistant models. ENDX combinations were predominantly additive in 2D assays, with limited synergy confined to specific dose windows. In 3D culture, selected combinations of an MCL1 inhibitor (MCL1i) or navitoclax with ENDX produced greater suppression than single agents and, in several conditions, reduced growth below baseline, consistent with loss of viability. Combination treatment also enhanced suppression of migration under select concentrations. In MCF7 xenograft studies, ENDX robustly suppressed tumor growth; however, adding MCL1i or navitoclax did not provide additional antitumor benefit at the doses, schedules, and timeframes tested. Conclusion: BH3 mimetics show activity in ERα+ breast cancer models in vitro , including endocrine resistant models. Although ENDX combination benefit did not translate in vivo under the conditions tested, these data support further study of optimized BH3 mimetic combinations, including evaluation of dose, ratio, schedule, tumor target engagement, and other clinically advanced agents.

Abstract Breast cancer is the most common female malignancy in the U.S. and the leading cause of cancer-related deaths in women worldwide. Nearly 80% of breast tumors express estrogen receptor alpha (ERα) which drives tumor progression. While endocrine therapies and CDK4/6 inhibitors have increased overall survival, many patients experience recurrence of metastatic disease that is largely refractory to additional lines of endocrine therapy. Metastatic disease remains incurable, highlighting the need for alternative treatment approaches. Through a genome-wide CRISPR knockout screen, we identified Lemur Tail Kinase 2 (LMTK2) as the top hit exhibiting synthetic lethality in the presence of the endocrine therapy, endoxifen, in both endocrine-sensitive and -resistant cell line models. Using publicly available patient tumor databases we identified LMTK2 copy number gain/amplification in 67% of ERα+ human metastatic breast tumors, while only 1% of primary tumors harbored these genomic alterations. Elevated LMTK2 expression in early-stage primary breast tumors was associated with worse recurrence-free survival and overall survival. We discovered that LMTK2 mRNA and protein levels were substantially elevated in nearly all cell-line and patient-derived models of endocrine and CDK4/6i resistance. Utilizing siRNAs and dox-inducible shRNA expressing cell lines, we found that knockdown of LMTK2 enhanced the efficacy of multiple endocrine therapies in treatment naïve models and resensitized resistant models to their respective treatment. LMTK2 overexpression was found to be sufficient for conferring resistance to all standard of care therapies tested including endocrine agents, CDK4/6i, and PI3K inhibitors. RNAseq and global proteomics of LMTK2 overexpressing and knockout models revealed the PI3K/AKT pathway and estrogen signaling as highly regulated pathways by LMTK2. Confirmatory western blot studies demonstrated that LMTK2 overexpression results in dramatic increases in AKT and ERα phosphorylation and activity, while knockout of LMTK2 diminishes these effects. Attempts to completely ablate LMTK2 in endocrine/CDK4/6i-resistant models via CRISPR were not successful, and continuous siRNA or shRNA-mediated knockdown was lethal in resistant cell lines, confirming its essentiality in this setting. In summary we have uncovered LMTK2 as a novel mediator of endocrine/CDK4/6i resistance that is mechanistically linked to the PI3K/AKT pathway among others. Genetic manipulation of LMTK2 has validated it as an ideal therapeutic target for both advanced and newly diagnosed forms of ERα+ breast cancer. Citation Format: Anna Detry, Sofia Marigliano, Jenna Heinen, Adam Bass, Calley Jones, Rajeev Muthyala, Matthew Goetz, John Hawse. LMTK2: A novel driver of endocrine resistance in ERα+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 443.

Abstract Breast cancer is the most common female malignancy in the U.S. and the leading cause of cancer-related deaths in women worldwide. Nearly 80% of breast tumors express estrogen receptor alpha (ERα) which drives tumor progression. While CDK4/6 inhibitors in combination with endocrine therapy improve overall survival in the metastatic setting and disease-free survival in the adjuvant setting, many patients eventually relapse/progress with resistant forms of disease. In the post-CDK4/6 inhibitor setting, second line endocrine therapy responses are limited, highlighting the need for alternative treatment approaches. Through a genome-wide CRISPR knockout screen, we identified Lemur Tail Kinase 2 (LMTK2) as the top hit exhibiting synthetic lethality in the presence of the tamoxifen metabolite, endoxifen, in both endocrine-sensitive and -resistant cell line models. We further studied multiple other endocrine resistant models and identified that LMTK2 mRNA and protein expression were both increased in ERα cell lines resistant to estrogen deprivation, tamoxifen, fulvestrant, abemaciclib and palbociclib, as well as in endocrine resistant patient derived models. We then studied LMTK2 in patient samples and identified copy number gain/amplification in 67% of ERα+ human metastatic breast tumors, while only 1% of primary tumors harbored these genomic alterations. From a prognostic standpoint, elevated tumoral LMTK2 expression was associated with worse recurrence-free survival and overall survival in early-stage breast cancer. Through proteomic studies, we identified the MET-PI3K-AKT pathway as overactivated in LMTK2 overexpressing cells. Unlike activating PIK3CA mutations that drive AKT signaling, LMTK2 was found to employ an alternative mechanism to activate AKT signaling by inhibiting PP1α, an AKT-inhibitory phosphatase. Further, LMTK2 overexpression was sufficient to drive endocrine and CDK4/6 inhibitor resistance in vitro and resulted in dramatic increases in AKT and ERα phosphorylation and activity. While knockout of LMTK2 in treatment naïve ERα+ breast cancer cells neither altered AKT/ERα phosphorylation/activity nor endocrine/CDK4/6 inhibitor responsiveness, attempts to knockout LMTK2 in endocrine/CDK4/6 inhibitor-resistant models failed, further confirming its essentiality in this setting. In summary we have uncovered LMTK2 as a novel biomarker of endocrine/CDK4/6 inhibitor resistance that is mechanistically linked to the AKT pathway. LMTK2 may be a novel drug target for the effective treatment of resistant and advanced forms of ERα+ breast cancer. Citation Format: A. M. Detry, S. Marigliano, J. Heinen, A. Bass, C. Jones, R. S. Muthyala, M. P. Goetz, J. R. Hawse. Lmtk2: a novel driver of endocrine resistance in ER+ breast cancer [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 PS2-11-01.

Abstract Estrogen receptor alpha (ERα)-positive breast cancer accounts for up to 80% of cases. Despite advances in CDK4/6 inhibitors (CDK4/6i) and endocrine therapies, recurrence and late relapse remain significant challenges. Recurrence often arises from disseminated cancer cells that persist despite 5-10 years of systemic adjuvant treatment. While CDK4/6i and endocrine therapies induce prolonged growth arrest resembling senescence, they do not significantly kill cancer cells. Senescent/pseudo-senescent cancer cells are implicated in aggressive variant formation, recurrence, and metastasis. We explored eliminating breast cancer cells via senolysis while they are in therapy-induced senescent state to prevent their potential to escape, repopulate, or cause long-term adverse effects. Live-cell imaging of MCF7 and T47D cells treated with abemaciclib and endocrine therapies (endoxifen or estrogen deprivation to mimic aromatase inhibition) at clinically relevant concentrations for eight days demonstrated prolonged growth cessation and G1 cell cycle arrest even two weeks post-drug withdrawal. These cells displayed a senescence-like gene expression profile, with increased CDKN2B (p15), CDKN1A (p21), BCL2, reduced KI67, and increased TGFβ expression, a known Senescence-Associated Secretory Phenotype (SASP) factor linked to deep senescence. These results confirm effective senescence induction by various CDK4/6i and endocrine therapy combinations. Further analysis using established senescence markers (e.g., nuclear size, telomere-associated foci, satellite distension, and loss of nuclear HMGB1 and LaminB1) revealed unique senescence phenotypes across therapy combinations. Cell viability assays and live imaging of Caspase 3/7 activation demonstrated differential vulnerabilities of senescent/senescent-like ERα+ cancer cells (MCF7, T47D, UCD4, UCD12) to 13 senolytics (1 μM), including natural flavonoids and inhibitors targeting pro-survival factors, tyrosine kinases, and the Notch pathway. For example, MCF7 and UCD4 cells responded to MCL1i and RSL3, T47D to BCLXLi and RSL3, and UCD12 to BCLXLi and Navitoclax. Notably, senolytic cocktails (e.g., RSL3, GNF7, Venetoclax, Dasatinib) at reduced concentrations (0.25 μM each) effectively eliminated senescent/senescent-like ERα+ cancer cells across all cancer cell lines, achieving 100% elimination in T47D cells. Collectively, these findings demonstrate that despite variability in therapy-induced senescence, senolytic combinations effectively target senescent ERα+ cancer cells. This provides preliminary evidence for the potential integration of this strategy into standard breast cancer therapy with a goal to improve recurrence-free survival and increase the cure rates for ERα+ breast cancer patients. In vivo studies are underway to evaluate this strategy in preclinical models. Citation Format: Lena Batoon, Angelo J. Guilatco, Anna Detry, Xiyin Wang, Megan M. Weivoda, John R. Hawse. CDK4/6 inhibitors and endocrine therapies induce senescence with drug- and cell line-specific vulnerabilities in ERα+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 519.