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Additional file 7: Fig. S5. Effect of PD-L1 and MT Oas2 on primary tumor initiation and expansion in parous mice. Examples of tumor growth curves of individual mice. The 2nd/3rd and 4th mammary glands were palpated twice weekly and tumor growth was estimated by measurement of the major and minor axis of individual tumors using calipers. Y axis is placed at the day of tumor detection on the x axis as day 0. Black diamonds show a treatment with IP IgG or PD-L1 antibodies and red diamonds indicate parity. WT = wild type, MT = MT Oas2 MT/MT = homozygous MT Oas2.

Additional file 8: Fig. S6. Effect of PD-L1 and MT Oas2 on primary tumor initiation and expansion in parous mice. Two-way Kaplan–Meier survival analysis for the indicated periods and endpoints, and for the indicated genotypes and treatments (PD-L1 or IgG IP), of mouse cohorts. P values and hazard ratios (HR) calculated by the log-rank test using GraphPad Prism. n = 8 WT PD-L1, 8 WT IgG, 14 MT PD-L1, 15 MT IgG.

Additional file 6: Fig. S4. Flow cytometry gating strategy for monocytes and neutrophils known as myeloid-derived suppressor cells. Series of gates used to quantify monocytes and neutrophils using flow cytometry.

Additional file 5: Fig. S3. Effects of MT Oas2 on T cells in parous mice. A screening of various T-cell parameters was undertaken in mammary tumors, lymph nodes, spleen and thymus. Error bars are standard error of the mean and p values calculated by Student’s t test (all non-significant). Axes represent % of total cells passing the previous gate. Genotypes, WT/WT homozygous wildtype Oas2, MT/MT homozygous mutant Oas2.

Additional file 1: Table S1. Antibodies, concentration and antigen retrieval conditions for immunohistochemistry. All reagents were from Leica BOND or DAKO for automated or manual IHC (as specified). Visualization of antigen–antibody complexes was performed using the DAB + liquid substrate chromogen system (K3467).

Additional file 3: Fig. S1. Effect of MT Oas2 on primary tumor expansion in parous mice. Examples of tumor growth curves of individual mice. The left- and right-hand side 2nd/3rd and 4th mammary glands were palpated twice weekly and tumor growth was estimated by measurement of the major and minor axis of each gland using calipers. Y axis is placed at parity indicated on the x axis as day 0. Numbers show the total number of glands with tumors detected, the number that showed a period of no growth, and the number that showed regression at or following parity. W, wild type; M, MT; W/W, homozygous wild type; M/M, homozygous MT.

Additional file 4: Fig. S2. Effect of MT Oas2 on primary tumor initiation and expansion in nulliparous and parous mice. Two-way Kaplan–Meier survival analysis for the indicated periods and endpoints, and for the indicated genotypes, WT wildtype Oas2, MT mutant Oas2, and parity status of mouse cohorts (20 per group). P values and hazard ratios (HR) calculated by the log-rank test using GraphPad Prism. n = 19 MT parous, 21 WT parous, 20 MT nulliparous and 19 WT nulliparous.)

Additional file 2: Table S2. Antibodies used for flow cytometry. Panel name, antibody name by antigen recognized and conjugated fluorochrome, the antibody supplier and catalogue number and final diluted concentration for each is shown.

During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mammary luminal progenitor cell. In breast cancer, ELF5 is a key transcriptional determinant of tumor subtype and has been implicated in the development of insensitivity to anti-estrogen therapy. In the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT) model of luminal breast cancer, induction of ELF5 levels increased leukocyte infiltration, angiogenesis, and blood vessel permeability in primary tumors and greatly increased the size and number of lung metastasis. Myeloid-derived suppressor cells, a group of immature neutrophils recently identified as mediators of vasculogenesis and metastasis, were recruited to the tumor in response to ELF5. Depletion of these cells using specific Ly6G antibodies prevented ELF5 from driving vasculogenesis and metastasis. Expression signatures in luminal A breast cancers indicated that increased myeloid cell invasion and inflammation were correlated with ELF5 expression, and increased ELF5 immunohistochemical staining predicted much shorter metastasis–free and overall survival of luminal A patients, defining a group who experienced unexpectedly early disease progression. Thus, in the MMTV-PyMT mouse mammary model, increased ELF5 levels drive metastasis by co-opting the innate immune system. As ELF5 has been previously implicated in the development of antiestrogen resistance, this finding implicates ELF5 as a defining factor in the acquisition of the key aspects of the lethal phenotype in luminal A breast cancer.

3073 Background: Comprehensive genomic profiling (CGP) is increasingly used to guide therapy selection in advanced cancer patients who have exhausted standard therapy options. Here we assess the utility of Therapy Oriented Precision Oncology Guidelines for Recommending Anticancer Pharmaceuticals (TOPOGRAPH) to guide matching of drug treatments based on CGP in this setting. Methods: This study was conducted in an Australia-wide precision oncology program, the Molecular Screening and Therapeutics Study (MoST, ANZCTR registration ACTRN12616000908437). All patients with advanced cancer after exhausting standard treatments underwent CGP in 2016-2021 were stratified into cohort A (no further therapy received) and B (received ≥1 therapy after CGP). The primary outcome was overall survival (OS) estimated by the Kaplan-Meier method, using the log rank test to assess between-group differences. TOPOGRAPH matched the treatment history to the CGP results, stratified into clinically active (Tiers 1-3, T1-3), investigational (T3B/4), inactive (R2) or unmatched groups. Results: Over a median follow-up of 21.7 months (mo) for 2852 patients (75% with rare cancers, n = 2150), the median OS (mOS) from the date of CGP result was 7.0 mo (95% CI 6.4-7.6) for cohort A (n = 1562) and 15.8 mo (95% CI 14.5-16.9) for cohort B (n = 1290). In both cohorts, patients with CGP results matching any TOPOGRAPH tier (T1-4) had shorter OS compared to patients without a matching tier (A: 6.4 v 20.5 mo, hazard ratio for death [HR] 2.15, p

Acquired resistance to endocrine therapy is responsible for half of the therapeutic failures in the treatment of breast cancer. Recent findings have implicated increased expression of the ETS transcription factor ELF5 as a potential modulator of estrogen action and driver of endocrine resistance, and here we provide the first insight into the mechanisms by which ELF5 modulates estrogen sensitivity. Using chromatin immunoprecipitation sequencing we found that ELF5 binding overlapped with FOXA1 and ER at super enhancers, enhancers and promoters, and when elevated, caused FOXA1 and ER to bind to new regions of the genome, in a pattern that replicated the alterations to the ER/FOXA1 cistrome caused by the acquisition of resistance to endocrine therapy. RNA sequencing demonstrated that these changes altered estrogen-driven patterns of gene expression, the expression of ER transcription-complex members, and 6 genes known to be involved in driving the acquisition of endocrine resistance. Using rapid immunoprecipitation mass spectrometry of endogenous proteins, and proximity ligation assays, we found that ELF5 interacted physically with members of the ER transcription complex, such as DNA-PKcs. We found 2 cases of endocrine-resistant brain metastases where ELF5 levels were greatly increased and ELF5 patterns of gene expression were enriched, compared to the matched primary tumour. Thus ELF5 alters ER-driven gene expression by modulating the ER/FOXA1 cistrome, by interacting with it, and by modulating the expression of members of the ER transcriptional complex, providing multiple mechanisms by which ELF5 can drive endocrine resistance., Author summary Two thirds of breast cancers are initially treated with endocrine therapy because they are likely to rely on estrogen for their proliferation. Understanding why this therapy ultimately fails in 2/3 of cases offers the chance of durable treatment. In 2012 we hypothesised that normal developmental cell fate decisions taken by mammary progenitor cells persist in tumours that are maintained by instances of a cancerous progenitor, and that a change in the relative influence of the two major transcription factors that drive progenitor cell fate, ER to specify the hormone sensing lineage, and ELF5 to specify the ER- alveolar lineage, may allow a cancer to shift control of proliferation from estrogen to ELF5. Here we show that these transcription factors are often co located at super enhancers, enhancers and at the promoters of differentially regulated genes. When the levels of ELF5 were increased this caused ER and its pioneer factor FOXA1 to move to new regions of the genome associated with resistance to hormonal therapy. We also showed that ELF both regulated and bound directly to members of the ER-transcriptional complex. These findings provide the first indication of the mechanisms by which an increase in ELF5 could drive the acquisition of endocrine resistance.