Minnesota Oncology

Pancreatic ductal adenocarcinoma (PDA) remains highly lethal, in part, because its dense fibroinflammatory stroma restricts therapy distribution, including adoptive T cell immunotherapies where direct interactions between T and carcinoma cells are essential for effective therapy. While T cell function must be maintained once effector-target engagement occurs, without inducing co-localization subsequent cytotoxic function steps cannot be undertaken. We therefore developed a strategy to "physically optimize" T cells to more effectively sample complex tumor volumes. Informed by pharmacologic perturbations and mathematical modeling we shifted T cell phenotype through expression of constitutively activated RhoA to increase cortical contractility, activation, migration, and sampling in PDA, while showing decreases in exhaustion markers. In CAR T cells this results in more efficient targeting through decreased sampling time and increased engagement with carcinoma cells, consistent with modeling predictions. This significantly increases T cell infiltration and distribution in PDA, resulting in improved tumor control in vivo, suggesting that this is an effective strategy to overcome stromal constraints, improve tumor engagement, and enhance the therapeutic performance of engineered T cell therapies in solid tumors.

ABSTRACT Individuals with Hereditary Breast and Ovarian Cancer (HBOC) syndrome carry BRCA1/2 mutations that predispose them to various cancers. Notably, this condition increases the risk of developing an aggressive ovarian cancer (OC) subtype known as high-grade serous ovarian cancer (HGSOC). The impact of progesterone (P4) on the development of OC subtypes remains unclear. Research suggests a potential interplay between Progesterone Receptor (PR) signaling and BRCA1/2 actions. BRCA1 regulates PR expression and transcriptional activity, while P4 can drive estrogen receptor/PR complexes to BRCA1 DNA binding motifs. Normal fallopian tube tissues from healthy BRCA1/2 carriers (the cells of origin for HGSOC) exhibit gene signatures similar to those in HGSOC only during the luteal phase of the menstrual cycle, when circulating P4 levels are elevated. This implies a potential role for PR/BRCA complexes in modulating OC initiation and progression. To explore this interaction, BRCA1 was depleted in an ES-2 PR-B+ OC cell models using CRISPR-mediated gene editing. We hypothesized that BRCA1 depletion would alter PR-mediated signaling and reveal novel regulatory mechanisms. Depletion of BRCA1 led to increased total basal PR protein levels and site-specific hyperphosphorylation at S294 and S81 following R5020 (synthetic progestin) treatment. RNA sequencing of NE-1 and KO-BRCA1 pools treated with R5020 identified a high-confidence set of BRCA1-dependent, R5020-induced genes predominately involved with migration and cytoskeletal organization. Transwell assays revealed that R5020 treatment stimulates migration, an effect amplified upon BRCA1 depletion. Inhibition of PIP5K1C using UNC3230 blocked R5020-induced migration in one of the KO-BRCA1 cell pools with pan-Rho inhibitor CT04 showing similar results. The Rac1 inhibitor EHT-1864 suppressed all R5020-induced migration. In contrast, co-treatment with a pan-ROCK1/2 inhibitor Y-27632 resulted in an increase in R5020-induced migration, which was further enhanced with BRCA1 depletion. Overall, these findings establish a crosstalk between BRCA1 and PR signaling, where BRCA1 depletion sensitizes OC cells to progestin-induced migration by altering PR phosphorylation and remodeling cytoskeletal organization via PIP5K1C, Rho-GTPase, and Rac1-dependent mechanisms.

Abstract BACKGROUND Mirdametinib is the first FDA-approved MEK1/2 inhibitor for both adults and children (≥2 years) with NF1 who have symptomatic PN not amenable to complete resection. We report updated results from the LTFU phase of the pivotal, phase 2b ReNeu trial (NCT03962543) evaluating the efficacy and safety of mirdametinib. MATERIALS AND METHODS In ReNeu, mirdametinib capsules or tablets for oral suspension (2 mg/m2 BID, max 4 mg BID) are administered in 3-weeks-on/1-week-off 28-day cycles. Following the 24-cycle (~2 years) treatment phase, patients could continue mirdametinib in an optional LTFU phase. These analyses are inclusive of the treatment and LTFU phases (data cutoff: June 12, 2024; an additional 9 months of cumulative data following the primary analysis). Exploratory analyses reported here included confirmed objective response rate (ORR; proportion of patients with ≥20% reduction of target PN volume from baseline on consecutive MRI scans within 2 to 6 months, assessed by blinded independent central review), duration of response (DoR), change from baseline in target PN volume, and safety. RESULTS Fifty-eight adults and 56 children were dosed with mirdametinib. Median (range) duration of mirdametinib treatment was 21.8 (0.4, 54.4) months in adults and 25.4 (1.6, 48.5) months in children. Among LTFU-eligible patients, 26/31 (84%) adults and 32/37 (86%) children entered the LTFU. Confirmed ORR was 47% (27/58; 95% CI, 33%, 60%) in adults and 55% (31/56; 95% CI, 42%, 69%) in children. Of patients with a confirmed objective response, 18 adults and 19 children achieved a deep response (best target PN volume reduction from baseline of >50%). Median (range) best percentage change from baseline in target PN volume was -41% (-90%, 13%) in adults and -43% (-98%, 48%) in children. Median DoR was not reached. Treatment-related adverse events (TRAEs) in ≥25% of patients were dermatitis acneiform, diarrhoea, nausea, and vomiting in adults, and diarrhoea, dermatitis acneiform, and paronychia in children. Grade ≥3 TRAEs occurred in 17% of adults and 25% of children. Dose interruptions, dose reductions, and discontinuations due to TRAEs occurred in 9%, 17%, and 22%, respectively, of adults, and in 14%, 14%, and 9%, respectively, of children. CONCLUSIONS An increase in confirmed ORR was observed with longer duration of mirdametinib treatment, with additional deep responses achieved in both adults and children with NF1-PN. Mirdametinib continued to be well tolerated with a manageable safety profile and no new safety signals identified in the LTFU phase. SUPPORTED BY SpringWorks Therapeutics, Inc.

ABSTRACTProstate cancer is the most common non‐cutaneous cancer among men in the United States. Most prostate cancers are driven by androgen receptor (AR) signalling, but there are an increasing number of cases that lose AR and gain neuroendocrine (NE) features (AR−/NE+) or lack both (AR−/NE−). These latter subtypes are particularly aggressive and lethal. Extracellular vesicles (EVs) have shown great potential as biomarkers for non‐invasive liquid biopsy assays, as EVs contain biomolecules from their cells of origin. Here, we used a shotgun proteomics approach with mass spectrometry to interrogate the global proteome of EVs isolated from prostate cancer cell lines reflecting diverse clinical subtypes, including AR−/NE+ and AR−/NE− models. We identified 3952 EV proteins, which clustered largely by tumour subtype and provided enough proteomic coverage to derive classic gene signatures of AR or NE identity that are of high relevance for prostate cancer prognostication. EVs isolated from AR+ cells displayed high levels of proteins regulated by AR and mTOR signalling. EVs isolated from AR−/NE+ cells contained known NE markers such as SYP and CHGA, whereas EVs from AR−/NE− models were enriched in basal cell markers and proteins that regulate epithelial‐to‐mesenchymal transition (EMT). We integrated our cell line data with recently published EV proteomics data from 27 advanced prostate cancer patients and found 2733 overlapping proteins, including cell surface markers relevant to prostate cancer, AR activity indicators, and proteins enriched in specific subtypes (AR+, AR−/NE−, AR−/NE+). This approach may be useful for rare cancer subtypes, such as prostate cancers that lose AR‐related features and gain NE features, to optimise the use of these liquid biopsy samples for clinical decision making.

Oncogenic fusions of MAML2 with CRTC1, CRTC3, YAP1, and NR1D1 retain the MAML2 transactivating domain (TAD) and are believed to drive aberrant gene transcription. While the oncogenic roles of these known fusions have been established, we aimed to identify novel MAML2 fusions across a range of human malignancies. DNA and RNA sequencing were performed on tumor samples submitted to Caris Life Sciences. MAML2 fusions were identified from RNA transcripts and filtered to include only known pathogenic fusions or recurrent, in-frame fusions containing a C-terminal MAML2 TAD. Fusion burden was defined as the number of unique fusion isoforms per sample. Among 180,124 tumor samples, 143 specimens harbored MAML2 fusions with a MAML2 TAD: >50% of specimens harbored known fusions, but novel fusions with MTMR2 (31/143), SESN3 (11/143), CCDC82 (6/143), FAM76B (4/143), and ATXN3 (3/143) were also identified. Compared to the known fusions, the novel fusions generally had lower expressions (median: 8 vs. 13 junction reads/sample, p = 0.0064), higher fusion burdens (median: 6 vs. 2 unique fusion isoforms/sample, p < 0.0001), more frequent TP53 co-mutations (80% vs. 11.5%, p < 0.0001), and no clear association with the tissue of origin. Excluding ATXN3::MAML2, the novel fusion partners were located near MAML2 in the genome, likely arose from duplications or deletions, and occurred in samples harboring concurrent mutations. In contrast, ATXN3::MAML2 arose via interchromosomal translocation, occurred in samples with a low fusion burden, and was not associated with TP53 mutations. We identified novel MAML2 fusion partners, most of which likely represent passenger alterations, possibly arising from genomic instability or impaired p53 function. However, ATXN3::MAML2 fusions, previously reported in a pre-cancerous pancreatic disease case, may represent a pathogenic alteration warranting further investigation.