Bass, Adam D.

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 Introduction: Immune checkpoint blockade (ICB) has clinical activity in triple negative breast cancer (TNBC) but is less effective in the ER+HER2- signature, where there is a cold immune microenvironment (IM) and regulatory T cells (Tregs) may suppress effector T cells. Agents that activate the IM by turning cold tumors hot may support ICB. The biguanides hexyl-benzyl-biguanide (HBB) and its bioisostere hexyl-(cuban-1-yl-methyl)-biguanide (HCB) are candidate agents to activate the IM because they potently inhibit biosynthesis of immunosuppressive epoxyeicosatrienoic acids (EETs) and EET-driven oxidative phosphorylation (OXPHOS), while blocking N-glycosylation of immune checkpoint (IC) proteins. We hypothesized that reversal of hypoxia by biguanides in the ovarian dependent ER+HER2- STAT1 KO SSM2ucd mouse mammary carcinoma (MC) model would suppress Tregs and promote effector T cells in the tumor IM. While the SSM2ucd model did not express immune checkpoint protein PD-L1 (B7-H1), it did express related IC proteins B7-H3 and B7-H4. We hypothesized that by inhibiting OXPHOS and reducing N-glycosylation of immune checkpoint proteins, HBB and HCB may promote efficacy of ICB. We chose the SSM2ucd model to test impact of HCB on the ER+ MC IM. Results: SSM2ucd cells exhibited longer tumor latency (60 days) than the basal 4T1 (10 days) and 67NR (20 days) mouse MC models. SSM2ucd tumor reimplantation shortened latency by more than half, to 20 days. Immunohistochemistry showed that B7-H3 and B7-H4 protein levels were 1.2 (P=0.001) and 1.3-fold (P=0.04) higher in reimplanted tumors vs. control. In SSM2ucd cells, HCB inhibited N-glycosylation of B7-H3 (P=0.01) by 35% and B7-H4 (P=0.02) by 45% and suppressed TGFβ induction of B7-H3 by 21% (P=0.02) and B7-H4 by 79% (P=0.001) at 24 hours, while 14,15-EET promoted N-glycosylation of B7-H3 (1.2-fold; P=0.03) and B7-H4 (1.3-fold; P=0.04) at 4 hours. Effects of HBB and HCB on anti-CD3 and anti-CD28 stimulated mouse splenocytes were assayed. The proliferative effects of HBB on CD4+ and CD8+ cells peaked at 12 uM (p<0.001) and for HCB at 6.25 uM (p<0.001). Tregs decreased with HCB at a threshold of 12 uM (p <0.001) and with HBB at 25 uM (P < 0.001) while 14,15-EET increased Tregs (1.2 fold; P=0.02) and suppressed the CD8+:Treg ratio (0.79; P=0.02). In the SSM2ucd mammary allograft model, although HCB 12mg/kg daily did not inhibit tumor growth, it reduced intratumoral hypoxia by 20% (P=0.01), increased CD8+ TIL by 4.4-fold (P=0.04), decreased Treg:CD4+ TIL ratio by 76% (P=0.02), and decreased Treg:CD8+ TIL ratio by 88% (P=0.01) relative to control. Conclusion: B7-H3 and B7-H4 expression inversely correlated with latency of ER+ MC and may represent targets for immune checkpoint antibodies and their drug conjugates. HCB, an inhibitor of OXPHOS and EET biosynthesis, reduced intratumoral hypoxia, increased CD8+ TIL and reduced the Treg:CD8+ ratio, potentially supporting ICB therapy of ER+ MC by turning cold tumors hot. Supported by CDMRP BCRP Grant BC180596, Award Number W81XWH-19-1-0099 Citation Format: Zhijun Guo, Jianxun Lei, Hrishi Venkatesh, David Owen, Adam Bass, Christine Cannon, Joshua McCarra, Brenda Koniar, Craig Flory, Beverly Norris, Robert J. Schumacher, Swaathi Jayaraman, John Hawse, Emmanuel S. Antonarakis, Emanuel F. Petricoin, Julia Wulfkuhle, Robert D. Cardiff, Elizabeth A. Ambrose, Gunda I. Georg, Kaylee L. Schwertfeger, Michael A. Farrar, Brad St. Croix, Matthew P. Goetz, David A. Potter. Hexyl-(cuban-1-yl-methyl)-biguanide (HCB) suppresses N-glycosylation of immune checkpoint proteins B7-H3 and B7-H4, reverses tumor hypoxia, decreases intratumoral regulatory T cells, and increases intratumoral CD8+ T cells in the ovarian dependent ER+HER2- SSM2ucd mammary cancer allograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB102.