DNA methyltransferase 1 (DNMT1) is essential for mammalian development and is frequently dysregulated in cancer. While its recruitment to hemimethylated DNA by UHRF1 is well established, its broader chromatin occupancy and regulation remain unclear. We show that DNMT1 is enriched at unmethylated CpG islands of actively transcribed genes largely by its CXXC domain. Upon selective catalytic inhibition, DNMT1 redistributes to partially methylated, inaccessible regions in a UHRF1-dependent and RFTS-dependent manner. Although coding gene expression is largely unchanged, sustained DNMT1 inhibition and subsequent global DNA hypomethylation trigger reactivation of endogenous viral elements, including an uncharacterized class of double-stranded RNA mega-intergenic transcripts (mintRNAs) driving a cell-intrinsic viral mimicry response. We identify RNF4 as a major regulator of DNMT1 catalytic function that, beyond its canonical role in DNA-protein crosslink resolution, controls the levels of SUMOylated DNMT1, establishing SUMOylation as a rheostat that regulates DNMT1 activity to maintain DNA methylation and safeguard against viral mimicry.

Somatic gene mutations (SGMs) that drive clonal haematopoiesis (CH) have been shown to be more prevalent in people with HIV (PWH), potentially contributing to the higher risk of comorbidities in PWH compared to those without HIV. It is unknown whether mosaic chromosomal alterations (mCA), another form of CH, are associated with HIV. We demonstrate, for the first time, a markedly lower prevalence of mosaic chromosomal alterations (mCA), particularly loss of chromosome Y, in PWH compared to participants without HIV - an opposing pattern to SGMs. Our findings that mCA development may be suppressed in HIV infection suggest that the selective pressures driving mCA-related CH differ fundamentally from those driving SGM-related CH.

1064 Background: PIK3CA mutations are a key therapeutic target in ER+ advanced breast cancer (ABC) readily detectable through circulating tumor DNA (ctDNA). The PI3Kα inhibitor alpelisib is approved in ER+ HER2- ABC but it remains unclear when to incorporate it into the current treatment algorithm. To address this, we conducted a phase II study assessing superiority of alpelisib + fulvestrant vs capecitabine in ABC pts with detectable PIK3CA mutant ctDNA following CDK4/6i plus aromatase inhibition (AI). Methods: Eligible pts had ER+ HER2- ABC, prior progression on CDK4/6i+AI and PIK3CA mutations detected via ctDNA ddPCR. Pts were randomized 1:1 to receive alpelisib (300mg daily) + fulvestrant (Arm A), or capecitabine (1000-1250mg/m 2 BD D1-14 q21d) (Arm B); strata were prior chemo for ABC and visceral disease. Primary endpoint was progression free survival (PFS) defined from randomization until progressive disease per RECIST 1.1 or death. Secondary endpoints included objective response rate (ORR), clinical benefit rate (CBR) and adverse events (AE). Imaging was q8 weeks. Exploratory objectives included efficacy according to baseline ctDNA levels. Sample size was reduced from 140 to 66 pts randomized for 53 PFS events to detect median PFS of 9 vs 5 mo (HR=0.56;1-sided α=0.1,80% power). Interim inefficacy analysis (60% events) estimated HR>1.0 and IDMC recommended early termination. Follow-up continued until all pts completed alpelisib. Reported are stratified Cox model HR (2-sided 80% CI) and 2-sided log rank test. Results: From July 2020-Oct 2024, 396 pts were screened, 113 (29%) had PIK3CA mutant ctDNA, 58 were randomized and 55 initiated treatment (Arm A: 29/30; Arm B: 26/28). Median age was 56 (32-82 yrs), 75% had visceral disease; 87% had no prior chemo for ABC. At final analysis, 43 PFS events occurred. Median PFS was 7.4 mo for alpelisib + fulvestrant vs 9.4 mo for capecitabine (HR 1.28, 80% CI: 0.84-1.96, p=0.45). For pts with visceral disease, median PFS was 5.4 mo (80% CI:3.7-5.1) for alpelisib + fulvestrant and 12.1 mo (80% CI:5.4-15) for capecitabine. ORR was 24.1% vs 50.0% and CBR 55.2% vs 61.5% for alpelisib + fulvestrant vs capecitabine. Overall, 79.3% pts on alpelisib + fulvestrant had grade 3/4 AEs with 24% grade 3 hyperglycemia vs 46.2% on capecitabine with 12% grade 3 palmar-plantar erythrodysesthesia. Six of 30 pts discontinued alpelisib due to AEs; 5 of the 6 continued fulvestrant. There was no evidence of association between baseline ctDNA levels (PIK3CA mutant copies/ml; median 90; range 2-13678) and PFS (HR 1.03; 90%CI:0.90-1.19). Conclusions: In our trial of pts with PIK3CA mutant ABC after progression on CDK4/6i plus AI, targeted therapy with alpelisib + fulvestrant was not superior to standard chemotherapy with capecitabine. Compared with the targeted therapy, capecitabine was better tolerated and resulted in longer PFS. Clinical trial information: ACTRN12619001117101.

Intratumoral heterogeneity (ITH) is fueling tumor progression in breast cancer, as specific clones present within a tumor may have a selective advantage to colonize distant organs and escape therapy. Accurate sampling of ITH is therefore a pressing challenge in clinical oncology to adequately predict recurrence and inform rational and personalized therapies. Here, we used genetic barcoding to track the spatiotemporal composition of human breast cancer clones in six preclinical models-across two cell lines and four patient-derived xenografts (PDXs). This allowed a direct side-by-side quantitative comparison of both intra-tumor clonal composition and how that composition was reflected in needle biopsies and cell-free DNA (cfDNA). These analyses highlighted several biologically and clinically relevant findings. First, the use of barcoding revealed that clonal diversity in the center of non-necrotic primary tumors was significantly higher than in the periphery. Second, cfDNA barcode analysis suggested that DNA 'shedding' in the vasculature varied widely, not only depending on necrosis and tumor burden but also across models. Third, combining information captured in both solid and liquid biopsies can provide a more robust assessment of tumor clonal composition. Taken together, these results showcase the utility of these barcoded models to optimize the use of solid and liquid biopsies as surrogates of tumor heterogeneity.

Several transcription inhibitors have been developed as cancer therapies. However, they show modest clinical activity, highlighting that our understanding of the cellular response to transcriptional inhibition remains incomplete. Here we report that potent inhibitors of transcription not only impact mRNA output but also markedly impair mRNA transcript localization and nuclear export. We demonstrate that retention of newly transcribed mRNA in nuclear speckles is an adaptive response to chemically distinct transcriptional inhibitors. Retained transcripts are fully processed and accumulate in proportion to the expression level of the genes from which they emanate. The TREX mRNA export complex plays an integral role in directing nascent transcripts to nuclear speckles where they are bound to NXF1, protected from degradation, and poised for rapid export following re-initiation of transcription. Our findings provide new insights into the crosstalk between transcription and mRNA export with important implications for drugs aiming to inhibit transcription for therapeutic gain.

Monitoring levels of circulating tumour‐derived DNA (ctDNA) provides both a noninvasive snapshot of tumour burden and also potentially clonal evolution. Here, we describe how applying a novel statistical model to serial ctDNA measurements from shallow whole genome sequencing (sWGS) in metastatic breast cancer patients produces a rapid and inexpensive predictive assessment of treatment response and progression‐free survival. A cohort of 149 patients had DNA extracted from serial plasma samples (total 1013, mean samples per patient = 6.80). Plasma DNA was assessed using sWGS and the tumour fraction in total cell‐free DNA estimated using ichorCNA. This approach was compared with ctDNA targeted sequencing and serial CA15‐3 measurements. We identified a transition point of 7% estimated tumour fraction to stratify patients into different categories of progression risk using ichorCNA estimates and a time‐dependent Cox Proportional Hazards model and validated it across different breast cancer subtypes and treatments, outperforming the alternative methods. We used the longitudinal ichorCNA values to develop a Bayesian learning model to predict subsequent treatment response with a sensitivity of 0.75 and a specificity of 0.66. In patients with metastatic breast cancer, a strategy of sWGS of ctDNA with longitudinal tracking of tumour fraction provides real‐time information on treatment response. These results encourage a prospective large‐scale clinical trial to evaluate the clinical benefit of early treatment changes based on ctDNA levels.

The mutational landscape of phylogenetically ultraconserved elements (UCEs), especially those in noncoding DNAs (ncUCEs), and their functional relevance in cancers remain poorly characterized. Here, we perform a systematic analysis of whole-genome and in-house targeted UCE sequencing datasets from more than 3000 patients with cancer of 13,736 UCEs and demonstrate that ncUCE somatic alterations are common. Using a multiplexed CRISPR knockout screen in colorectal cancer cells, we show that the loss of several altered ncUCEs significantly affects cell proliferation. In-depth functional studies in vitro and in vivo further reveal that specific ncUCEs can be enhancers of tumor suppressors (such as ARID1B) and silencers of oncogenic proteins (such as RPS13). Moreover, several miRNAs located in ncUCEs are recurrently mutated. Mutations in miR-142 locus can affect the Drosha-mediated processing of precursor miRNAs, resulting in the down-regulation of the mature transcript. These results provide systematic evidence that specific ncUCEs play diverse regulatory roles in cancer.

Genomics can inform both tissue-of-origin (TOO) and precision treatments for patients with cancer of unknown primary (CUP). Here, we use whole genome and transcriptome sequencing (WGTS) for 72 patients and show diagnostic superiority of WGTS over panel testing (386-523 genes) in 71 paired cases. WGTS detects all reportable DNA features found by panel as well as additional mutations of diagnostic or therapeutic relevance in 76% of cases. Curated WGTS features and a CUP prediction algorithm (CUPPA) trained on WGTS data of known cancer types informs TOO in 71% of cases otherwise undiagnosed by clinicopathology review. WGTS informs treatments for 79% of patients, compared to 59% by panel testing. Finally, WGS of cell-free DNA (cfDNA) from patients with a high cfDNA tumour fraction (>7%), enables high-likelihood CUPPA predictions in 41% of cases. WGTS is therefore superior to panel testing, broadens treatment options, and is feasible using routine pathology samples and cfDNA.

Background: CDK4/6 inhibitors induce growth arrest/senescence in breast cancer cells, which become refractory to apoptotic cell death. In preclinical models, the BCL2 inhibitor venetoclax augmented tumor response to endocrine and CDK4/6 inhibitor therapy, by triggering apoptosis. This is now being tested in PALVEN, a phase 1b dose escalation study in patients (pts) with ER+ and BCL2+ metastatic breast cancer (MBC). The maximum tolerated and recommended phase 2 dose has been defined as letrozole 2.5 mg (d1-28), palbociclib 75 mg (d1-21) and venetoclax 400 mg (d1-21). Here, we present the clinical benefit rate (CBR) and response rate (RR) at 24 wks and report treatment-related toxicity. Methods: Participants included post-menopausal women with ER+/HER2– (ASCO/CAP) and BCL2+ MBC who had received ≤2 prior lines of systemic therapy in the metastatic setting. Patients (pts) with evaluable disease were included. Tumor assessment occurred every 8 wks. Secondary endpoints included CBR and RR defined by RECIST v1.1 within the first 24 wks of treatment. Adverse events from venetoclax, palbociclib and letrozole were reported using the CTCAE v5.0 grading system. Results: As of 19 June 2024, sixteen pts had received venetoclax, palbociclib and letrozole treatment and fifteen were DLT-evaluable (median age 50 yrs [range 36-67]), with a median follow-up time of 29.2 months. Of the DLT-evaluable population, 8 pts had de novo MBC and 7 pts had relapsed MBC, with 9 pts having previously received endocrine therapy in either the adjuvant or metastatic setting. Three pts had received prior systemic treatment for MBC; 1st line aromatase inhibitor therapy (2 pts), chemotherapy followed by tamoxifen (1 pt). At the 24-wk tumor assessment, 60% (9/15) of patients had achieved a partial response (PR), 33% (5/15) had stable disease (SD), and one pt had progressive disease. The CBR (rate of confirmed complete response or PR or SD ≥24 wks) was 93% (14/15 pts). Of thirteen pts with baseline measurable disease, 9 (69%) achieved a PR by 24 wks. In pts who were previously untreated in the metastatic setting, the CBR was 100% (12/12), 89% (8/9) for pts who had previously received endocrine therapy in the adjuvant or metastatic setting. For pts with strong (IHC 3+) BCL2 expression, CBR was 88% (7/8). An interim safety analysis of all eligible pts that received at least one dose of triple therapy (n=16), determined the most common non-hematological treatment-related adverse events (TRAEs) of any grade were fatigue (75%), nausea (69%), arthralgia (50%), hot flashes (44%), constipation (44%) and diarrhea (44%). Grade ≥3 hematological TRAEs were decreased neutrophil count (63%), reduced white cell count (44%) and decreased lymphocyte count (19%). There were no AEs of special interest since dose level 1 (G3/4 AST/ALT elevations in 2 pts). No treatment-related deaths were reported. Two pts ceased triple therapy due to intolerable TRAE, including one with arthralgia attributed to letrozole alone. Conclusion: Triple therapy with palbociclib, letrozole and venetoclax in ER+ and BCL2+ MBC is well tolerated and shows promising clinical activity in pts with endocrine sensitive and CDK4/6 inhibitor-naive disease. Our findings suggest that combining a BCL2 and CDK4/6 inhibitor in endocrine responsive breast cancer merits further investigation. (IIS funded by NHMRC, NBCF, Cancer Australia, BCRF, BCT, VCCC, with support from AbbVie & Pfizer; NCT03900884). Citation Format: Geoffrey Lindeman, Christine Muttiah, Felicity C Martin, Michael Christie, Sarah-Jane Dawson, Jayesh Desai, Luxi Lal, Kate Moodie, Anand Murugasu, Phuong Phan, Mark R Rosenthal, Avraham Travers, Jane E Visvader, James R Whittle, Belinda Yeo. Results from PALVEN: A Phase 1b Study of Palbociclib, Letrozole and Venetoclax in ER and BCL2-Positive Metastatic Breast Cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P2-07-18.

The use of circulating tumour DNA (ctDNA) to profile mutational signatures represents a non-invasive opportunity for understanding cancer mutational processes. Here we present MisMatchFinder, a liquid biopsy approach for mutational signature detection using low-coverage whole-genome sequencing of ctDNA. Through analysis of 375 plasma samples across 9 cancers, we demonstrate that MisMatchFinder accurately infers single-base and doublet-base substitutions, as well as insertions and deletions to enhance the detection of ctDNA and clinically relevant mutational signatures. Accurately detecting cancer mutational profiles from circulating tumour DNA (ctDNA) remains a challenging task. Here, the authors develop MisMatchFinder, an algorithm that can detect mutational signatures in shallow whole-genome sequencing data from ctDNA across multiple cancer types.

Abstract Background: Advanced HER2-positive breast cancer remains an incurable and highly morbid condition. One of the key challenges has been treatment of central nervous system (CNS) metastases, commonly seen in those with HER2-positive disease. Tucatinib is a recently approved treatment HER2-targetting tyrosine kinase inhibitor with proven CNS activity. There has been little analysis of potential genomic mechanisms of drug resistance. Method: We retrospectively identified patients that were treated with tucatinib, capecitabine and trastuzumab. We collated baseline clinical data and treatment history and all tumour and plasma samples available were obtained for testing. DNA was extracted from plasma samples using the QIAamp DNA Mini Kit (Qiagen) on 4ml plasma samples. Targeted next generation sequencing (NGS) of serial tumour and plasma samples was performed using a custom hybrid capture assay that covered 180 genes known to be recurrently mutated in breast cancer focusing on the known genomic landscape of metastatic disease as well as the inclusion of specific actionable targets. Survival analyses was performed using Cox regression models. Results: We identified 11 patients at our institution. Plasma samples were available for all 11 patients with a total of 107 serial samples across multiple time points, with a median of 8 (range 2-19) samples per patient. All 11 patients had baseline and End of Treatment (EOT) samples. In addition, 6/11 (54%) patients had tumour sequenced on the same NGS platform prior to commencement of therapy, with 12 individual tumour samples tested. The median PFS of our cohort was 9.6 months (4.0 - NR) with a median OS of 36.2 months (10.4-NR). 7/11 (64%) patients had CNS disease at baseline with their median PFS being 13.5 months (3.9-24.4) and median OS of 36.2months (10.4-NR). Overall, we observed an average of 18.4 (4-35) mutations per patient in tumour, 5.1 (1-15) in baseline plasma and 5.8 (2-14) in EOT plasma. The most frequent mutations seen in baseline tumour samples were ERBB2 (92%), KMT2C (67%) and NCOR1 (58%) compared with KMT2C (64%), NOTCH4 (36%) and PIK3CA (27%) in baseline plasma. Patients with a lower number of ctDNA mutations at baseline showed a trend towards improved survival (PFS 14 months vs 7.5 months). In this small cohort of patients we identified a higher than expected rates of KMT2C mutations. A KMT2C mutation was identified in 67% of samples which is higher than previously described rates of 7-15% in TCGA and METABRIC cohorts respectively. The presence of a concordant ctDNA KMT2C mutation in baseline and progression samples was a poor prognostic indicator (mPFS 7.5 months) when compared with no KMT2C mutation (mPFS 15.5 months) p =0.14 suggesting a possible mechanism of resistance to tucatinib. Conclusions: In this retrospective analysis of patients treated with Tucatinib, Trastuzumab and Capecitabine we established the genomic landscape of heavily pre-treated, advanced HER2-positive disease is complex. We observed there may be a correlation with KMT2C mutations and poorer clinical outcomes, however given the very small number of patients this data remains hypothesis generating, requiring validation with larger datasets. Citation Format: Elizabeth Blackley, Courtney van Geelen, Yi-An Ko, Stephen Wong, Miriam Yeung, Stephen Luen, Sarah-Jane Dawson, Sherene Loi. Investigation of the genomic evolution of HER2-positive breast cancer following progression on dual HER2-targetted therapy with Trastuzumab and Tucatinib [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-04-06.

ABSTRACT:In the phase 2 clinical trial (AIM) of venetoclax-ibrutinib, 24 patients with mantle cell lymphoma (MCL; 23 with relapsed/refractory [R/R] disease) received ibrutinib 560 mg and venetoclax 400 mg both once daily. High complete remission (CR) and measurable residual disease negative (MRD-negative) CR rates were previously reported. With median survivor follow-up now exceeding 7 years, we report long-term results. Treatment was initially continuous, with elective treatment interruption (ETI) allowed after protocol amendment for patients in MRD-negative CR. For R/R MCL, the estimated 7-year progression-free survival (PFS) was 30% (95% confidence interval [CI], 14-49; median, 28 months; 95% CI, 13-82) and overall survival (OS) was 43% (95% CI, 23-62; median, 32 months; 95% CI, 15 to not evaluable). Eight patients in MRD-negative CR entered ETI for a median of 58 months (95% CI, 37-79), with 4 experiencing disease recurrence. Two of 3 reattained CR on retreatment. Time-to-treatment failure (TTF), which excluded progression in ETI for those reattaining response, was 39% overall and 68% at 7 years for responders. Beyond 56 weeks, grade ≥3 and serious adverse events were uncommon. Newly emergent or increasing cardiovascular toxicity were not observed beyond 56 weeks. We demonstrate long-term durable responses and acceptable toxicity profile of venetoclax-ibrutinib in R/R MCL and show feasibility of treatment interruption while maintaining ongoing disease control. This trial was registered at www.clinicaltrials.gov as #NCT02471391.