Yan, Yiyi

Abstract Effector gene expression in CD8+ T cells is tightly controlled at the epigenetic and transcriptional levels; however, crucial regulatory signals remain incompletely understood. In this study, we identify dual specificity phosphatase 2 (DUSP2) as a key epigenetic modulator of effector CD8+ T-cell state, by elucidating inhibition of BAF-mediated chromatin opening by DUSP2. Induced downregulation of DUSP2 in human CD8+ T cells enhances cytotoxicity and effector phenotypes by increasing chromatin accessibility through upregulation of BAF subunits, such as SMARCA4, and permitting binding of ETS1 to BAF subunit gene loci and those essential for effector programs. These findings uncover a previously unrecognized role of DUSP2 in the management of the chromatin landscape in effector CD8+ T cells and the epigenetic fine-tuning of their functionality, highlighting DUSP2 as a promising therapeutic target for T cell–based immunotherapy.

Abstract Understanding how immune checkpoint inhibitors (ICIs) reshape human T-cell responses requires models that move beyond static transcriptomic snapshots and discrete cell-state classifications. Here, we present an integrative AI-enabled virtual cell framework that represents human CD8⁺ T-cell responses as dynamic and computable systems during ICI therapy. By integrating single-cell RNA sequencing with paired T-cell receptor sequencing within the C2S-scale foundation model, we construct a virtual representation of individual T cells, in which each cell is encoded by a unique functional identity that captures its transcriptional, signaling, and clonal characteristics. Using this framework, we identify a previously unrecognized dynamic latent effector state of CD8⁺ T cells characterized by intermediate expression of effector genes, distinct signaling activity, and ongoing clonal expansion. Across independent patient cohorts, the virtual cell model consistently indicates that ICI therapy mainly acts by unmasking pre-existing effector potential rather than inducing de novo effector differentiation. Notably, this latent effector population remains transcriptionally restrained despite active signaling and clonal expansion, revealing a hidden reservoir of antitumor immune capacity. More broadly, our study demonstrates how AI-enabled virtual cell modeling can reconstruct latent cellular states and their dynamic transitions from multidimensional single-cell data. By incorporating functional identity into virtual cell model, this framework uncovers biologically meaningful yet non-obvious T-cell effector program during cancer immunotherapy and provides a generalizable approach for studying immune dynamics in human disease.

Abstract Description CD8 T cell programming creates a pool of naïve, effector, memory, or exhausted subsets, however the regulation of these trajectories is not expansively elucidated and has serious implications for cancer immunity and immune checkpoint inhibitor (ICI) therapy. We found a significant decrease of Dual Specificity Phosphatase 2 (DUSP2) in CX3CR1+ CD8s, a highly resilient subset, from ICI responders. Thus, we hypothesized that DUSP2 negatively regulates CD8 T cells in responses to ICI therapy. DUSP2 knockdown (KD) statistically improved CD8 cytotoxicity and expression of NKG7, a vital effector molecule. DUSP2 KD also increased SMARCA4 expression, a BRG/BRM-associated factor (BAF) complex ATPase, and increased open chromatin regions of BAF components. BAF complexes are chromatin remodelers implicated in CD8 differentiation pathways. In line with this, we found CD8s from DUSP2 conditional knockout (cKO) mice demonstrated a trend in defective memory generation. DUSP2 cKO CD8s also trended towards decreased PD-1 and increased CX3CR1 expression, demonstrating a more effector like phenotype. DUSP2 cKO mice did not show a significant difference in tumor protection in-vivo, presumably due to skewing of CD8 fates via BAF. Promisingly, DUSP2 KD increased ex-vivo cytotoxic functionality in about 50 percent of tested cancer patient PBMCs. Adjuvant BAF inhibition with DUSP2 KO versus KD in a specific CD8 subset may enhance responses further, providing a novel avenue for improving ICI efficacy. Funding Sources Supported by NCI 1F31CA287495-01 Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

e21554 Background: Pregnancy associated melanoma (PAM) occurs in 1% of female patients with melanoma and affects 1 in 2200 pregnancies. Antepartum evaluation of melanoma must incorporate both maternal and fetal factors, and currently there is lack of standardization. Our study aimed to review the incidence of antepartum melanoma at our institution and highlight patterns in management and treatment during this high-risk scenario. Methods: After IRB approval, the Mayo Clinic electronic medical record was searched with key words “melanoma” and “pregnancy”. 25 patients with melanoma during pregnancy were identified from 1987-2023. 6 patients were excluded as skin changes noted during pregnancy were not formally diagnosed as melanoma until after delivery. Abstracted data included demographic information, disease characteristics, staging (per AJCC guidelines at time of diagnosis), and clinical outcomes. Survival data were calculated. Results: 19 patients were included. Diagnosis occurred at an average maternal age of 30 years (range 24-38 years) and a mean gestational age of 20 weeks (range 6-35 weeks). All patients had a term delivery. 4 patients underwent labor induction at 37 weeks for staging/treatment. 60% of patients had a delay in imaging or treatment attributed to pregnancy, including 1 patient who was upstaged with metastatic disease based on postpartum imaging. 82% of stage I-III patients underwent wide local excision during pregnancy; however, 35% of sentinel lymph node sampling was delayed until postpartum. 3 stage II/III patients received adjuvant therapy (interferon or sargramostim) after delivery. Adjuvant immunotherapy was discussed with 2 patients in the postpartum period but ultimately not given due to the time lapse between surgery and delivery. 1 patient with stage IV disease diagnosed at a gestational age of 20 weeks was evaluated with MRI PET scan then treated with vemurafenib monotherapy during pregnancy. Workup and outcomes were reviewed by stage (Table 1). 7 of 19 patients had subsequent pregnancies, and 1 had recurrence of melanoma during a second pregnancy (stage II followed by stage IV). Due to a low number of censoring events, median progression free survival and overall survival were not reached. Conclusions: Melanoma diagnosed during pregnancy can be safely and effectively staged with sentinel lymph node surgery, MRI imaging (PET-MRI vs standard), and CT imaging with shielding without a need for early induction of labor nor a delay in cancer care. Furthermore, we report a patient with stage IV disease treated with vemurafenib during and immunotherapy after pregnancy, indicating that treating stage IV PAM with systemic therapy is a consideration. [Table: see text]

e23270 Background: Melanoma is the most lethal form of skin cancer which have seen drastic improvement in patient survival over the last decade. BRAF is the most common driver mutation in melanoma, seen in ≈50% of all patients (≈30% of patients > 65). The use of BRAF & MEK inhibitors yields high response rates, though it is limited by toxicity and development of acquired resistance. Acknowledging these limitations, we aimed at assessing the efficacy and toxicity of these agents in melanoma patients > 65. Methods: We identified electronic medical records of all melanoma patients > 65 that were treated with BRAF & MEK inhibitors at our institution over the years 2017-2023. Data was retrospectively collected including demographics, disease characteristics, treatment given, response patterns and toxicity. Results: 106 patients were identified. The median age was 71 (65-89), 55% were males. The median follow up was 28.9 months. 32 (30%) patients were treated at first line, 58 (55%) at second line and 16 (15%) at third line. 88 (83%) evaluable patients received treatment for unresectable/metastatic disease of which 84% had an objective response (60% partial response, 24% complete response). Response rate (RR) was 82%, 85% and 87% for first, second and third line respectively. RR was 86% for patients with an increased serum lactate dehydrogenase. 33% of patients had active central nervous system (CNS) metastases at treatment initiation, for whom the systemic RR was 80% and the CNS RR was 39%. The median progression free survival (PFS) was 7.9 months, with a PFS of 6, 8.3 and 13.4 months for first, second and third line respectively. The median PFS was 5.7 months for patients with active CNS disease. The median overall survival (OS) from treatment initiation was 15.7 months (12.5, 14.5, 31.9 for first, second & third line respectively). The median OS for patients with CNS disease was 9.8 months. 16 patients were treated in the adjuvant setting, of which 6 (37%) finished treatment as planned, 3 stopped due to disease progression (19%) and 6 stopped due to toxicity (37%). 88% of patients experienced adverse events (AEs) of any grade, with 27% having grade 3-4 toxicity. There were no grade 5 toxicities reported. 36% of patients experienced one AE, while 52% experienced two or more. 21% of patients stopped treatment due to toxicity, 28% needed a dose reduction and 20% changed to different BRAF/MEK inhibitors. 10% of patients needed systemic corticosteroids to treat AEs. The most common toxicities seen were fever (30%), fatigue (30%), skin rash (22%), elevated liver function tests (20%), arthralgia/myalgia (15%), nausea (14%) and diarrhea (11%). 7% had decrease in cardiac ejection fraction with one grade 3 leading to treatment discontinuation. Conclusions: Treatment with BRAF & MEK inhibitors is challenging in older patients, with a high prevalence of toxicity leading to treatment modifications and discontinuation. Efficacy seems to be the same as in the general population.

e23269 Background: Most cases of malignant melanoma are cutaneous, with ≈50% having a BRAF driver mutation that predicts response to MAPK pathway inhibitors. A small minority of melanoma cases harbor an activating KIT mutation, most notably in mucosal and acral melanomas (15-20%) and occasionally in those arising from chronic sun-damaged skin (2-3%). Only about a third of KIT mutated melanomas respond to targeted therapy with Imatinib, yet data is scarce and limited, especially in older patients. We sought to characterize the efficacy and toxicity of Imatinib in KIT-mutated melanoma patients over 65. Methods: We identified electronic medical records of patients over 65 that were treated with Imatinib for KIT mutated melanoma at our institution over the years 2017-2023. Data was retrospectively collected including demographics, disease characteristics, response patterns and toxicity. Results: 10 patients were identified, 50% were males. The median age at treatment initiation was 75 years (71-100). 3 patients had cutaneous primary, 3 had Vulvar/vaginal primary, 3 had acral primary and 1 had anal canal primary. All patients had normal Lactate dehydrogenase at treatment initiation and ECOG performance status was 0-2 (median 0). 9 patients received Imatinib for stage IV disease with an overall response rate (ORR) of 55% (44% partial response, 11% complete response). Median duration of response was 4 months. Responses were seen in acral melanoma (67%), vulvar/vaginal melanoma (67%) and cutaneous melanoma (33%). Median Progression free survival was 4.3 months (1.8-44), median overall survival from treatment initiation was 22.6 months (4-64). 6 patients were treated as 2nd line with an ORR of 67% and 3 were treated as 3rd line with an ORR of 33%. There was one patient with M1d disease who did not respond to treatment either systematically or intracranially. 6 patients had Exon 11 mutations (V560D/G/E, N564S and L576P) of which 4 responded (67%). 2 had Exon 17 mutations (N822C/K) of which 1 responded (50%) and the 2 others had an Exon 14 (F681I) and Exon 13 (K642E) mutation that did not respond to treatment at all. Toxicity was moderate with 90% of patients experiencing any grade AE, yet only 1 experienced toxicity requiring dose interruption with two Grade 3 toxicities - Pneumonitis and decreased neutrophil count, leading to temporary treatment interruption, dose reduction and a course of corticosteroids. The rest of the patients experienced only Grade 1-2 toxicity. 4 patients needed dose reductions, there were no treatment discontinuations due to toxicity. Conclusions: Treatment with Imatinib for elderly KIT mutated melanoma patients is safe and well tolerated with no treatment discontinuations recorded. The response rate is encouraging yet seems to vary according to the KIT mutation present as previously reported. The duration of response is usually short-lived and alternative/combination treatments are still needed for this patient population.

Abstract Background: The prognosis of patients with MM with CNS mets is poor. E6201 is an intravenous (IV) MEK inhibitor with activity against BRAF V600-mutated MM cell lines, including MAPK resistance alteration. A previous phase I trial demonstrated activity of E6201 in BRAF V600E mutated MM with a Maximal Tolerated Dose of 320mg/m2 given IV once a week x 3 weeks q 28 days. 2/23 (8.7%) of patients had a PR, 8/23 (34.8%) had SD. Two patients with MM and brain mets achieved near-CR which led to preclinical studies that demonstrated E6201 is unaffected by P-gp and BCRP transport and achieved higher brain to blood concentration (266%).In this trial, patients with BRAF mutation + MM with brain mets, single agent E6201 given at 320mg/m2 twice weekly x 3 weeks q 28 days was determined to be safe. One patient had CNS SD and a PR in lung mets, and a second patient achieved CNS stable disease and CR in a leg melanotic lesion. The drug was well-tolerated with one reversible AE thrombocytopenia (Gr 2). The final review showed intracranial response of SD in 2/4 (50%) patients and systemic SD in 3/4 (75%) patients. Methods: We are currently accruing to the dose escalation cohort of E6201 with dabrafenib 150 mg twice daily orally (n=up to 22 evaluable patients). E6201 is administered IV at 320mg/m2 twice weekly for 3 weeks every 28 days. Patients must have stage IV melanoma with BRAF mutation with at least one active lesion in the brain. Prior local therapy is allowed if done at least 3 weeks prior to cycle 1. Participants must be on a stable dose of steroids for at least 7 days and have an ECOG <!–=2. The study allows one prior BRAF/MEK inhibitor therapy. The primary objectives are to determine the MTD of E6201 when combined with dabrafenib and the response rate of brain mets. The secondary objectives include duration of response, extracranial response, progression free survival, overall survival, and impact of BRAF mutation subtype and safety of the combination. Clinicaltrials.gov: NCT05388877 References:1.Gampa G, et al. Brain distribution of a novel MEK inhibitor E6201: implications in the treatment of melanoma brain metastases. 2018. Drug Metab Dispos.2.Tibes R et al. Safety, pharmacokinetics, and preliminary efficacy of E6201 in patients with advanced solid tumors, including melanoma: results of a phase I study. 2018. Brit J of Cancer. 3.Babiker Gm et al, E6201, an intravenous MEK1 inhibitor, achieves an exceptional response in BRAF V600E mutated metastatic malignant melanoma with brain metastases. 2018. Inv New Drugs4.Gampa G, Kim M, Cook-Rostie N, Laramy JK, Sarkaria JN, Paradiso L, DePalatis L, Elmquist WF. Brain distribution of a novel MEK inhibitor E6201: Implications in the treatment of melanoma brain metastases. 2018. Drug Met Disp.–> Citation Format: Mahesh Seetharam, Roxana Dronca, Arkadiusz Z. Dudek, Garth Nelson, Matthew Block, Skylar Starling, Amber Baskin, Renee Bradshaw, Courtney Nelson, Kylee Andrews, Alisha Birgin, Cristina Watson, Yiyi Yan, Daruka Mahadevan, Svetomir Markovic, Tom Myers, Linda Paradiso, Hani Babiker. Phase 1 trial of MEK1 inhibitor E6201 plus dabrafenib in patients (pts) with BRAF V600-mutated metastatic melanoma (MM) with central nervous system (CNS) metastases (mets) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT157.

Abstract Purpose: AB160 is a 160-nm nano-immunoconjugate consisting of nab-paclitaxel (ABX) nanoparticles noncovalently coated with bevacizumab (BEV) for targeted delivery into tissues expressing high levels of VEGF. Preclinical data showed that AB160 resulted in greater tumor targeting and tumor inhibition compared with sequential treatment with ABX then BEV. Given individual drug activity, we investigated the safety and toxicity of AB160 in patients with gynecologic cancers. Patients and Methods: A 3+3 phase I trial was conducted with three potential dose levels in patients with previously treated endometrial, cervical, and platinum-resistant ovarian cancer to ascertain the recommended phase II dose (RP2D). AB160 was administered intravenously on days 1, 8, and 15 of a 28-day cycle (ABX 75–175 mg/m2, BEV 30–70 mg/m2). Pharmacokinetic analyses were performed. Results: No dose-limiting toxicities (DLT) were seen among the three dose levels tested. Grade 3/4 toxicities included neutropenia, thromboembolic events, and leukopenia. DL2 (ABX 150 mg/m2, BEV 60 mg/m2) was chosen as the RP2D. Seven of the 19 patients with measurable disease (36.8%) had confirmed partial responses (95% confidence interval, 16.3%–61.6%). Pharmacokinetic analyses demonstrated that AB160 allowed 50% higher paclitaxel dosing and that paclitaxel clearance mirrored that of therapeutic antibodies. Conclusions: The safety profile and clinical activity of AB160 supports further clinical testing in patients with gynecologic cancers; the RP2D is DL2 (ABX 150 mg/m2, BEV 60 mg/m2).

Patients with advanced cancers who either do not experience initial response to or progress while on immune checkpoint inhibitors (ICIs) receive salvage radiotherapy to reduce tumor burden and tumor-related symptoms. Occasionally, some patients experience substantial global tumor regression with a rebound of cytotoxic CD8 + T cells. We have termed the rebound of cytotoxic CD8 + T cells in response to salvage therapy as T cell resilience and examined the underlying mechanisms of resilience. Resilient T cells are enriched for CX3CR1 + CD8 + T cells with low mitochondrial membrane potential, accumulate less reactive oxygen species (ROS), and express more malic enzyme 1 (ME1). ME1 overexpression enhanced the cytotoxicity and expansion of effector CD8 + T cells partially via the type I interferon pathway. ME1 also increased mitochondrial respiration while maintaining the redox state balance. ME1 increased the cytotoxicity of peripheral lymphocytes from patients with advanced cancers. Thus, preserved resilient T cells in patients rebound after salvage therapy and ME1 enhances their resiliency.