Menin scaffolds the oncogenic histone-lysine-N-methyltransferase (KMT2A)-fusion protein (FP) complex in KMT2A-r and wild-type KMT2A complex in NPM1-m acute myeloid leukemia (AML). Menin inhibitors (MIs) are effective in KMT2A-r AML and NPM1-m AML. However, not all patients respond to MIs as monotherapy. In this preclinical study, we demonstrate that the MI ziftomenib, in combination with the XPO1 inhibitor selinexor, synergistically inhibited the growth of multiple KMT2A-r and NPM1-m AML cell lines (CI<1). The combination suppressed colony formation in primary CD34+ KMT2A-r progenitor cells without affecting normal stem cells. Robust apoptosis and decreased G2/M populations were also evident. The combination downregulated HOXA9 and MEIS1 while upregulating monocytic differentiation marker CD11b in both the AML molecular signatures. RNA sequencing and proteomic analysis in KMT2A-r revealed suppression of multiple bona fide menin-KMT2A target genes. Our mechanistic studies also identified a novel role of XPO1 in stabilizing menin's binding to chromatin and its interactions with KMT2A and KMT2A/MLLT3. XPO1 inhibitor-mediated disruption of these interactions, particularly in combination with ziftomenib, synergistically impairs oncogenic transcriptional programs. In vivo, combination therapy improved survival in both MV4;11 and OCI-AML3 cell line and primary patient-derived KMT2A-r and NPM1-m AML xenograft models in NSG mice, effective even at reduced drug doses. These preclinical findings demonstrate that simultaneous inhibition of the menin-KMT2A interaction and XPO1 can be a more effective translational strategy for treating KMT2A-r and NPM1-m AML than MI monotherapy to deepen responses and delay/prevent relapses.

Abstract A crucial aspect of the bioinformatics workflow in small RNA-sequencing is the alignment of reads to a database of reference ncRNAs. Alignment algorithms such as Bowtie, Burrows-Wheeler Aligner (BWA), and Spliced Transcripts Alignment to a Reference (STAR) - which are designed for aligning reads to a reference genome - are typically used. Aligning short RNA-sequenced reads to a database of non-coding RNAs (ncRNAs) is fundamentally a different task than aligning longer reads to a genome due to ncRNAs (i) having roughly the same number of nucleotides as the reads being aligned and (ii) being subsequences of other ncRNAs. To account for these differences, we developed the novel alignment algorithm LevenMap . Of all reads which exactly matched a reference ncRNA in a publicly available dataset, LevenMap aligned 100.0% of them to their respective ncRNA while all other aligners mapped less than 40% of these reads to their corresponding ncRNA. Furthermore, the mean ratio (length of read) / (length of corresponding reference ncRNA) of all aligned reads was 1.0 and 0.998 for LevenMap with at most zero and one mismatch(es) allowed, respectively; this ratio was no more than 0.51 for all other aligners. Overall, LevenMap is designed to account for the nuances of aligning small RNA-sequencing data to a database of reference ncRNAs and yields more biologically relevant counts compared to traditional aligners in this context. LevenMap is free and publicly available on GitHub: https://github.com/hdlugas/LevenMap .

Endometrioid adenocarcinoma has one of the strongest body mass index associations of any solid tumor. This is widely attributed to amplification of estrogen-driven signaling via increased production of unopposed estrogen in peripheral adipose tissue. However, there is little evidence as to whether this is a pure dosage effect or if persistent signaling leads to feedback on the execution of that signaling. Using single-nucleus RNA sequencing of primary tumors from postmenopausal patients with normal and obese body mass indices, we identified coordinated transcriptional remodeling across tumor epithelial, immune, and stromal compartments. Network-level analysis of tumor epithelial cells revealed that ESR1-associated co-expression modules in tumors from patients with obesity were distinct across two independent patient cohorts and transcriptomic modalities. These network constructions were then shown to be enriched for estrogen receptor targets using existing chromatin immunoprecipitation data from endometrioid adenocarcinoma cell lines and normal endometrial tissue, as well as RNA-seq following exogenous exposure to estrogen for the cell lines. Thus, this study provides a single-nuclei atlas of endometrioid adenocarcinoma and suggests that obesity likely both amplifies estrogenic signaling and qualitatively reshapes its regulatory context.

Antibody and IFN-γ responses and co-stimulatory and co-inhibitory marker expressions in mCRPC patients.

Dysregulated nuclear protein transport, commonly observed in cancer, results in the mislocalization-mediated inactivation of many proteins. We showed that overexpression of exportin 1 (XPO1) is linked to higher grade and Gleason score in metastatic castration-resistant prostate cancer (mCRPC). Using network topology computational approaches, XPO1 and PARP1 were identified as synthetic lethal partners and we have demonstrated synergy between XPO1 and PARP inhibitors in mCRPC cell lines. This study evaluates the combination’s efficacy in cell line-derived (CDX) and patient-derived xenograft (PDX) models, investigating the mechanisms of synergy. For the CDx and PDx models, 22rv1 cells and CTG-3581 tissue (Champions Oncology) were grown subcutaneously in ICR-SCID and CEIA/NOG male mice, respectively. XPO1 inhibitor was dosed orally at 10-15 mg/kg twice a week and PARP inhibitor was dosed orally at 50 mg/kg daily. For in vitro mechanistic study, 22rv1 cells were subjected to RNAseq, proteomic analysis, and Digital Spatial Profiling (DSP; 10x Genomics Visium platform) after treatment. Data analysis was performed using iPathwayGuide (advaitabio.com) and cLoupe browser. The XPO1-PARP inhibitors combination showed enhanced anti-cancer efficacy in both CDx and PDx models, with no significant weight loss or organ toxicity such as liver, spleen, and kidney. Survival benefits were evident in the combination group. Immunohistochemistry revealed apoptotic cell death, shown by cleaved caspase 3 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Mechanistic studies with 22rv1 cells showed downregulation of DNA replication genes (e.g., MCM6 and CDC6) and reduced enrichment scores for DNA replication pathways in gene set enrichment analysis. The proteomic analysis highlighted decreased levels of DNA replication modulators HMGB2 and DNAJC9. Besides the suppression of various DNA damage response genes (e.g. BRCA2 and RAD51), XPO1 inhibition also reduced the PARylation of mCRPC cells. DSP analysis of 22rv1 CDx tissue revealed a reduction of unbiased cellular clusters and increased differentially expressed genes in the combination treatment. The DNA replication pathway was among the top 5 pathways modulated by XPO1i treatment in DSP analysis. Taken together, this study revealed the therapeutic potential of XPO1-PARP inhibitors combination via targeting of the DNA damage response pathway in mCRPC. Md. Hafiz Uddin, Aaban A. Azmi, Laiba M. Monir, Amro Aboukameel, Husain Y. Khan, Sahar F. Bannoura, Frank Cackowski, Yusra Shao, Vinod Shidham, Sunil Jaiman, Rafic Beydoun, Gregory Dyson, Seongho Kim, Yang Shi, Vy Ong, Julie Boerner, Ramzi M. Mohammad, Elisabeth I. Heath. XPO1 inhibition synergizes with PARP inhibitor in metastatic castration resistant prostate cancer via the regulation of DNA damage response [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 5716.

Quiescent, or G0 cell cycle phase, cells are common in most prostate cancer (PCa) cases, yet are relatively resistant to many treatments, especially cytotoxic chemotherapy. They also play key roles in tumorigenesis, and dormancy and recurrence after curative intent therapy, but are difficult to identify when viable. To identify characteristics of quiescent prostate cancer cells, we used previously published FACS methods to isolate cancer cells from bone or bone marrow of eight metastatic castration resistant prostate cancer (mCRPC) patient samples and generated single cell RNA sequencing (scRNA-seq) gene expression data of individually sorted cells. We also generated scRNA-seq data from six PCa cell lines and two normal (cancer free) bone marrow samples as positive and negative controls for cell identification. We eliminated a priori any potentially contaminating cells based on tSNE cluster location, expression of a 12 gene panel of PCa markers, and lack of expression of bone marrow markers (PTPRC / CD45, and GYPA / CD235a), leaving 297 high confidence PCa cells. To identify characteristics of G0 cells, we separated the patient PCa cells into quiescent and cycling groups based on expression of MKI67 (Ki67). GSEA analysis of KEGG pathways revealed enrichment of primarily immune related pathways in quiescent cells with the top 3 pathways; “allograft rejection, ” “autoimmune thyroid disease, ” and “intestinal immune network for IgA production.” Similarly, for GSEA analysis using gene ontology (GO) groups, “beta-2-microglobulin binding” was the second most enriched group in quiescent cells. Type 1 HLA genes, were common among multiple pathway groups suggesting potential differences in antigen presentation. In subsequent validation experiments, induction of quiescence in LNCaP, C4-2B, PC3, and RM1 PCa cell lines increased cell surface HLA class 1 or Mhc-1. Overall, these results suggest that antigen presentation is increased in quiescent PCa cells, which may be important for their elimination by the immune system, or useful for future immunotherapy approaches. Kristina G. Ibrahim, Julie Boerner, Steven Zielske, Frank C. Cackowski. Single cell analysis reveals upregulation of immune stimulatory pathways in quiescent metastatic castration resistant prostate cancer cells [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 5097.

Over the past several decades, the incidence of endometrial cancer has risen. This trend is largely attributed to the increased prevalence of obesity and is especially pronounced in endometrioid adenocarcinoma, the most common histologic subtype of uterine cancer. These cancers are regarded as hormonally driven tumors and are linked to obesity through the production of aromatase, an enzyme that converts androgens into estrogen, in peripheral adipose tissue. Though the association between obesity and endometrial cancer is well-established, whether excess adiposity has an effect outside of this mechanism—and exactly how that hormonal imbalance perturbs the tumor and its microenvironment—is not well understood. Thus, to examine the impact of obesity on the cellular diversity and transcriptome of endometrial cancer and its microenvironment, we integrated single-nuclei RNA-seq data from seven obese and six normal-weight patients' endometrioid tumor samples. Through evaluation of marker gene expression patterns, we annotated seven clusters distributed across normal-weight and obese tumor samples. To pinpoint major changes in biological networks in tumor cells from obese and normal-weight patients, we performed single-nucleus pathway gene ontology analysis on differentially expressed genes. We then independently conducted Weighted Gene Correlation Network Analysis on the cancer cells in our normal and obese groups and compared the resultant networks. Our analysis revealed a large number of differentially expressed genes in both our cancer cluster and macrophage subcluster. In the cancer cluster, most of the differentially regulated genes were associated with estrogen signaling and epithelial-mesenchymal transition. The results from our Weighted Gene Correlation Network Analysis revealed large differences in the networks containing estrogen receptors. To model obesity in vitro, we treated 12Z endometrioid cells and KLE endometrioid adenocarcinoma cells with adipose-conditioned media derived from human omental tissue, which revealed large differences in cellular morphology. In our macrophage subcluster, our gene ontology analysis yielded differences in macrophage organization and function. Few tumor-infiltrating lymphocytes were detected, which is consistent with the classification of most endometrial cancers as immunologically “cold.” Overall, our results show that obesity influences gene expression in the endometrioid adenocarcinoma tumor microenvironment and tumor itself. Furthermore, the outcomes of our experiments provide additional insights into the impact of obesity on endometrioid adenocarcinoma outside of its role in estrogen signaling. Jessica L. Long, Sophia Agrusa, Swornalata Pukhrambam, Areebah Qazi, Katherine Gurdziel, Julie Boerner, Michael Wilson. Single nuclei sequencing reveals altered gene expression and immune signaling in tumors from obese endometrioid adenocarcinoma patients [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 1358.

Patient-derived xenograft (PDX) models are invaluable tools for studying the heterogeneity of primary human tumors and preclinical drug responses. However, traditional PDX models for estrogen receptor-positive (ER+) breast cancer face notable challenges. These models involve implanting tumor tissue into mice with the aim of achieving tumor "engraftment, " a process that typically requires 2 to 6 months and often longer for the tumor to grow to a size adequate for passaging. This extended timeframe, combined with the relatively indolent and less aggressive nature of hormone receptor-positive (HR+) breast cancers, results in lower success rates for tumor engraftment and passage. Additionally, these slow-growing tumors are prone to clonal selection during long-term growth, further reducing the utility of traditional PDX models for timely therapeutic studies. To address these challenges, we developed a streamlined PDX model optimized for HR+ breast cancer. Tumors were resected from deidentified patients at Karmanos Cancer Institute meeting specific eligibility criteria: ER or PR-positive, HER2-negative, and no prior neoadjuvant or endocrine therapy. Tumor specimens were aseptically implanted into the mammary fat pads of female SCID mice, which were supplemented with slow-release estradiol pellets to maintain estrogen levels. Tumor growth was monitored using caliper measurements, and tumor presence and vascularization were confirmed at 14 days post-implantation using Susceptibility Weighted Imaging (SWI). Following confirmation of tumor establishment, mice were randomized into two groups: a Tamoxifen-treated cohort (100 mg/kg orally for three days) and an untreated control group. Tumors were subsequently harvested for molecular and histological analyses. Our preliminary findings indicate that tumor engraftment and vascularization can be reliably achieved within two weeks, supporting the feasibility of this approach for time-sensitive therapeutic studies. Importantly, tumors remained present following treatment, providing adequate samples for downstream analyses after the experiment. This model circumvents many limitations of traditional PDX systems, including slow tumor growth and clonal selection, while providing a practical platform for studying endocrine therapy responses in HR+ breast cancer. By minimizing reliance on long-term engraftment and optimizing conditions for short-term assessment, this refined system holds promise for advancing translational research and improving preclinical evaluations of HR+ breast cancer therapies. Abigail M. Fielder, Manohar Ratnam, Lisa Polin, Julie Boerner, Seongho Kim, Kristen Purrington. Optimizing in vivo establishment of hormone receptor-positive breast cancer patient-derived xenografts for therapeutic assessment [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 47.

Waterfall plot showing PSA change from baseline to week 10.

Overall survival post-sipuleucel-T by race.

Black race and TLR1-602I SNP associate with stronger responses to TLR1/2 stimulation. A, Supernatant cytokine release separated by Black (n = 15) vs. White (n = 91) for each stimulation normalized to fold mock treatment control in the PRIME study; (*) Mann–Whitney test P < 0.05. B, TLR1-602 genotypes for each patient with sufficient DNA (n = 88 White; n = 15 Black) were tested by PCR-RFLP; percentages of I/I or I/S vs. S/S by race are shown. C, Mean fold mock control cytokine induction by genotype is shown for each stimulus; asterisks indicate significant FDR-corrected unpaired t tests Q < 0.05 (two-tailed). D, Mean fold mock control TLR1/2 induced cytokine induction by genotype for White vs. Black (all I/I or I/S); FDR-adjusted unpaired t test Q < 0.05 vs. White S/S (*) or vs. all other groups (#). E, Heatmaps depict −log (P values) from comparisons in B (cytokine induction by race) or C (cytokine induction by TLR1 SNP status) for each cytokine and treatment. See Supplementary Fig. S6 for extended data.

Co-stimulatory and co-inhibitory marker expression on CD4+ and CD8+ T cells in AA (n=29) and non-AA (n=28).

Summary of significant responses before and after sipuleucel-T in mCRPC patients.

Cytokine and chemokine responses in AA (n=29) and non-AA(n=28).

Th1 and Th2 cytokine and chemokine responses in mCRPC patients.

Co-stimulatory and co-inhibitory marker expression on CD4+ and CD8+ T cells in two racial groups. A, No differences in the percentage of CD4+ T cells were observed at baseline or at 10 weeks post-treatment in AA men (n = 29) vs. non-AA men (n = 28). Expression of co-stimulatory receptor ICOS on CD4+ T cells was significantly higher at baseline and at 10 weeks post-treatment (P = 0.0006; P = 0.0027, respectively) in AA men compared to non-AA men. Expression of co-inhibitory receptor BTLA on CD4+ was also significantly higher at baseline (P < 0.0001) in AA men compared to non-AA men. B, The percentage of CD8+ T cells was significantly lower (P = 0.0016) at baseline as well as at 10 weeks post- treatment in AA men compared to non-AA men. Expression of co-stimulatory receptor ICOS on CD8+ T cells was significantly higher at baseline and at 10 weeks post-treatment (P = 0.0023; P = 0.0026, respectively) in AA men vs. non-AA men. Differences between the two racial groups were analyzed by the Mann–Whitney test and P values < 0.0032 were considered as significant in the multiple comparisons sense. The horizontal line in each dot plot marks the median value. Base, baseline; wks, weeks.

The risk of developing subsequent breast cancer is higher in women diagnosed with benign breast disease (BBD) but these studies were primarily performed in non-Hispanic white populations. Still, these estimates have been used to inform breast cancer risk models that are being used clinically across all racial and ethnic groups. Given the high breast cancer mortality rates among African American (AA) women, it is critical to study BBD in this population, to ensure the risk models that include this information perform adequately. This study utilized data from AA women who underwent benign breast biopsies at a hospital served by the University Pathology Group in Detroit, Michigan, from 1998 to 2010. Patients were followed for subsequent breast cancers through the population-based Metropolitan Detroit Cancer Surveillance System (MDCSS). BBD lesion scores were assigned to represent the severity or extent of benign breast lesions, with higher scores indicating a greater number of distinct lesion types. Of 3,461 eligible AA women with BBD in the cohort, 6.88% (n=238) subsequently developed breast cancer. Examined individually, six of the eleven lesions (apocrine metaplasia, ductal hyperplasia, lobular hyperplasia, intraductal papilloma, sclerosing adenosis, columnar alterations and radial scars) were significantly associated with increased risk of breast cancer after adjustment for age and year of biopsy and were further considered in multiple lesion models. For every different type of benign breast lesion, subsequent risk of breast cancer increased by 25% (RR=1.25, 95% CI: 1.10, 1.42) after adjustment for age at biopsy and proliferative versus non-proliferative disease. In summary, this study affirms the increased breast cancer risk in AA women with BBD, particularly in those with multiple lesions. These findings have implications for the management of breast cancer risk in millions of women affected by BBD, a high risk group that could benefit from personalized surveillance and risk reduction strategies.

Introduction Clonal hematopoiesis of indeterminate potential (CHIP) is associated with an increased risk of cardiovascular disease, higher all-cause mortality, and elevated likelihood of developing hematological malignancies. Prior studies reported CHIP at a higher prevalence in solid cancer patients (~25-30%), possibly due to increased exposure to mutagenic/epigenetic stressors inherent in this population, common risk factors shared between solid cancers and CHIP or the oncogenic therapies they receive. Environmental factors may influence the epigenetic landscape, impacting the progression of CHIP to myeloid neoplasms (MN), including AML. Despite many studies, there is little known about the patterns of CHIP/CCUS among BAA and other minority groups. By assessing critical mutations in precursor conditions, we aim to improve health outcomes by preventing their clonal progression, given their poorer prognosis in overt disease stages. Notably, BAA AML patients have historically low survival rates due to socioeconomic, healthcare disparities, and biological factors. We here conducted a population sequencing study in our catchment area to study the prevalence of CHIP lesions in a racially distinct solid cancer population. Methods 76 adult female cancer patients (breast, endometrial, ovarian, uterine) with a history of cardiometabolic disease (Hx of HTN, HLD, DM, PVD, CAD) who were treated at Karmanos Cancer Institute with curative intent for solid cancer were sampled at their diagnosis prior to exposure to cytotoxic chemo or radiotherapy. The study was supported under the overarching proposal of ACHIEVE GreatER: Addressing Cardiometabolic Health Inequities by Early PreVEntion in the Great LakEs Region (NIH/NIMHD Grant #5P50MD017351). Race/ethnicity data was self-reported. Whole exome sequencing was used to identify CHIP mutation and single-nucleotide polymorphism (SNP) array data. A variant calling pipeline with Genome Analysis ToolKit was used to identify somatic variants, including variant quality score recalibration method for quality filtering and minimum variant allele frequency (VAF) of 0.02. Putative somatic CHIP mutations were further filtered using a list of commonly identified CHIP genes based on prior literature. Likely germline variants, defined as single nucleotide variants with VAF > 0.3, were excluded. Based on the literature, the following were exceptions and counted as somatic: DNMT3A R882, SRSF2 P95, SF3B1 K666 and K700, JAK2 V617. Chi-square and t-tests were used for univariate statistical analyses; logistic regression was used for multivariate analysis. Results Among the total cohort, the median age was 63 (IQR 56-68 ys). 35 (46%) patients had breast, 22 (29%) had endometrial, 14 (18%) had ovarian, 1 (1%) patient had cervical, 1 (1%) had uterine cancer, and 3 (4%) had benign endometrial/ovarian/cervical disease. The known cardiometabolic association was seen in 54% of patients, and a majority of patients had risk factors for coronary artery disease, including hypertension (84%), diabetes (42%), hyperlipidemia (37%), and smoking (39%). 45 patients (59%) had a known family history of malignancy. 45 (59%) patients were BAA, 25 (33%) were Non-BAA, and 6 (8%) were other races. After filtering germline variants, we focused our analysis on somatic mutations comparing disparities between BAA and Non-BAA patients. There was numerical enrichment of IDH2MT in BAA patients (9% vs. 3% in non-BAA, p=0.3). Notably, KMT2C mutation was significantly enriched in BAA patients compared to non-BAA (67 vs 23%, p=0.0002) and was independently associated with age (p<0.05), race (p=0.003), and known cardiac disorder (p<0.05). There were notable differences in other myeloid genes between races, including GATA2, STAG2, NPM1, DNMT3A, and others that were not statistically significant due to low sample size. In multivariate logistic regression analysis, both race and age were important factors in determining KMT2C mutation status (Non-BAA race, OR=0.20, p=0.009; age OR=1.07, p=0.051), and cardiac disorder trended toward KMT2C association (OR=2.82, p=0.062). Conclusion Our study is one of the first to evaluate the racial disparity in CHIP prevalence among solid cancer patients. Given the prognostic relevance of the presence of IDH2/1 mutations in overt disease stages like AML, these enriched mutations in BAA sets a premise for intervention to prevent clonal evolution.

Immune-centric Pathway Analysis of Genetic Predictors of PD-L1 Expression and TLS Status in NSCLC Cases.

Statins, inhibitors of HMG-CoA reductase, have been shown to have potential anti-carcinogenic effects through the inhibition of the mevalonate pathway and their impact on Ras and RhoGTAases. Prior studies have demonstrated a reduction in breast tumor proliferation, as well as increased apoptosis, among women with early-stage breast cancer who received statins between the time of diagnosis and the time of surgery. The aim of this study was to evaluate the impact of short-term oral high-potency statin therapy on the expression of markers of breast tumor proliferation, apoptosis, and cell cycle arrest in a window-of-opportunity trial. This single-arm study enrolled 24 women with stage 0-II invasive breast cancer who were administered daily simvastatin (20 mg) for 2–4 weeks between diagnosis and surgical resection. Pre- and post-treatment tumor samples were analyzed for fold changes in Ki-67, cyclin D1, p27, and cleaved caspase-3 (CC3) expression. Out of 24 enrolled participants, 18 received statin treatment and 17 were evaluable for changes in marker expression. There was no significant change in Ki-67 expression (fold change = 1.4, p = 0.597). There were, however, significant increases in the expression of cyclin D1 (fold change = 2.8, p = 0.0003), p27 cytoplasmic (fold change = 3.2, p = 0.025), and CC3 (fold change = 2.1, p = 0.016). Statin treatment was well tolerated, with two reported grade-1 adverse events. These results align with previous window-of-opportunity studies suggesting a pro-apoptotic role of statins in breast cancer. The increased expression of markers of cell cycle arrest and apoptosis seen in this window-of-opportunity study supports further investigation into the anti-cancer properties of statins in larger-scale clinical trials.