In many cancers, stably elevated MYC levels drive sustained activation of anabolic programs and the cell cycle, creating opportunities for the synthetic-lethal targeting of MYChigh tumors. Enhanced mitochondrial respiration is a hallmark of MYC overexpressing cancer cells. Mitochondrial respiration sustains the TCA cycle by regenerating NAD+ through complex I-mediated oxidation of NADH, supporting the anabolic demand of MYC-driven cells. Metabolic carbon tracing reveals that MYC shifts the TCA cycle carbon source from glucose to glutamine. Inhibition of the glutamine-fueled TCA cycle using NAD+-depleting complex I inhibitors promotes MYC-dependent synthetic lethality in breast cancer cells. In mouse models of MYChigh tumors, combined inhibition of complex I and glutaminolysis produces persistent suppression of tumor growth. Altogether, the elevated respiration of MYChigh cells supports a glutamine carbon-enriched TCA cycle that meets anabolic demand, rendering MYChigh tumors selectively vulnerable to mitochondrial respiration and glutaminolysis inhibitors.

This paper studies the problem of computing the stochastic probability (shortest code length) of the encoded vectors containing cluster structure using Normalized Maximum Likelihood (NML) model. This is of great theoretical and practical importance in data clustering based on Minimum Description Length (MDL) principle, such as for estimating the best number of clusters and best cluster structure for the data. Straightforward computation of the shortest code length of the vector containing cluster structure based on the NML model requires polynomial time with respect to the size of the vector and number of clusters. We show that this is a tractable problem by introducing a recursion formula for the efficient computation of normalizing constant from the NML model. The time complexity of the new formula is linear opposed to previous polynomial time with respect to the size of the vector and number of clusters.

Tumor-resident immune cells play a crucial role in eliciting anti-tumor immunity and immunomodulatory drug responses, yet these functions have been difficult to study without tractable models of the tumor immune microenvironment (TIME). Patient-derived ex vivo models contain authentic resident immune cells and therefore, could provide new mechanistic insights into how the TIME responds to tumor or immune cell-directed therapies. Here, we assessed the reproducibility and robustness of immunomodulatory drug responses across two different ex vivo models of breast cancer TIME and one of renal cell carcinoma. These independently developed TIME models were treated with a panel of clinically relevant immunomodulators, revealing remarkably similar changes in gene expression and cytokine profiles among the three models in response to T cell activation and STING-agonism, while still preserving individual patient-specific response patterns. Moreover, we found two common core signatures of adaptive or innate immune responses present across all three models and both types of cancer, potentially serving as benchmarks for drug-induced immune activation in ex vivo models of the TIME. The robust reproducibility of immunomodulatory drug responses observed across diverse ex vivo models of the TIME underscores the significance of human patient-derived models in elucidating the complexities of anti-tumor immunity and therapeutic interventions.

Oncogenic MYC promotes cancer cell proliferation, metabolism, and death, while also driving immunosuppression in the tumour microenvironment, complicating immune-based therapies. To counter MYC-driven immune evasion while leveraging MYC-dependent synthetic lethality (MYC-SL), we identified microtubule-targeting agents, including eribulin, as potent inducers of immunogenic cell death in MYChigh triple-negative breast cancer (TNBC). A screen of 528 oncology compounds using damage-associated molecular pattern (DAMP) reporters revealed that microtubule inhibitors induced key DAMPs, including HMGB1 secretion, calreticulin exposure, and double-stranded DNA release, leading to gasdermin-E associated cell death in MYChigh TNBCs. Immune cell co-culture assays showed immune activation, and patient-derived explant cultures confirmed pro-inflammatory cytokine responses. In vivo, cell-free media from eribulin-treated MYChigh murine TNBCs enhanced tumour protection in vaccination models compared to MYC-knockdown controls, linking MYC-dependent DAMP release to immunogenicity. These findings highlight a dual-function therapeutic strategy: agents that selectively induce MYC-dependent immunogenic cell death can provide both targeted cytotoxicity and local immune stimulation, thereby addressing a key limitation of conventional chemotherapeutics, offering a new approach for MYC-driven cancers. ### Competing Interest Statement The authors have declared no competing interest. Academy of Finland https://ror.org/05k73zm37, 350393 Business Finland https://ror.org/05bgf9v38, 2141/31/2019 Cancer Society of Finland https://ror.org/05pgg4z49 Sigrid Jusélius Foundation https://ror.org/00ckakm23, 240095 Jane and Aatos Erkko Foundation https://ror.org/03vxy9y38, 200035 European Union’s Horizon 2020, 847912 Department of Defence, Breast Cancer Research Program, CDMRP W81XWH211-0773/-0774 iCAN - Digital Precision Cancer Medicine Flagship Finnish Red Cross Blood Service Research Fund

Abstract Oncogenic MYC is frequently overexpressed in breast cancer, and its overexpression is associated with aggressiveness and unfavorable prognosis of breast cancer. MYC overexpression promotes tumor growth by stimulating cell cycle progression and rewiring metabolism – creating cancer vulnerabilities that can be therapeutically targeted via synthetic lethal strategies. A large part of our current understanding of the MYC overexpression biology and related synthetic lethal pathways is based on findings in various inducible MYC overexpression systems, which demonstrate the acute effects of MYC activation but may poorly represent long-term MYC overexpression status in real cancers. Here we classified 15 triple-negative breast cancer (TNBC) cell lines according to both MYC target gene set expression and MYC protein expression into MYC-high and MYC-low categories and explored the MYC-high status-coupled therapeutic responses and cancer-relevant metabolic changes. We show that mitochondrial respiration inhibiting biguanide drug metformin inhibits cell proliferation and generates senescence-like features in MYC-high TNBC cells. Furthermore, high-resolution respirometry experiments show that MYC-high TNBC cells have higher mitochondrial respiration rates than MYC-low TNBC cells, a phenotype not explained by a higher number of mitochondria. Also, targeting mitochondrial respiration with small molecule inhibitors specifically reduces proliferation in MYC-high TNBC cells and sensitizes them to apoptosis – indicating that MYC-high TNBC cells have higher dependency on mitochondrial respiration than the MYC-low cells. Targeted metabolomic flux experiments demonstrate that small molecule inhibitors of mitochondrial respiration rewire TCA cycle, resulting in suppression of MYC-driven glutamine anaplerosis, which offers a possible explanation for the apoptotic sensitization. In conclusion, our findings provide new insight into the long-term MYC overexpression-associated metabolic changes – highlighting mitochondrial respiration as a potential target for the development of MYC-dependent synthetic lethal strategies against breast cancer. Citation Format: Johanna M. Anttila, Mariel Savelius, Daniel Nicorici, Ryan Awadhpersad, Pauliina M. Munne, Linda Id, Topi A. Tervonen, Christopher Jackson, Anni I. Nieminen, Juha Klefström. Targeting MYC-induced reprogramming of mitochondrial metabolism in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A027.

Supplementary Table 6 from Systematic Analysis of MicroRNAs Targeting the Androgen Receptor in Prostate Cancer Cells

Supplementary Figures 1-2, Materials, Tables 1-4 and Table 7 from Systematic Analysis of MicroRNAs Targeting the Androgen Receptor in Prostate Cancer Cells

Supplementary Table 5 from Systematic Analysis of MicroRNAs Targeting the Androgen Receptor in Prostate Cancer Cells

BACKGROUND:Prostate cancer (PCa) lacks non-invasive specific biomarkers for aggressive disease. We studied the potential of urinary extracellular vesicles (uEV) as a liquid PCa biopsy by focusing on the micro RNA (miRNA) cargo, target messenger RNA (mRNA) and pathway analysis.METHODS:We subjected uEV samples from 31 PCa patients (pre-prostatectomy) to miRNA sequencing and matched uEV and plasma EV (pEV) from three PCa patients to mRNA sequencing. EV quality control was performed by electron microscopy, Western blotting and particle and RNA analysis. We compared miRNA expression based on PCa status (Gleason Score) and progression (post-prostatectomy follow-up) and confirmed selected miRNAs by quantitative PCR. Expression of target mRNAs was mapped in matched EV.RESULTS:Quality control showed typical small uEV, pEV, RNA and EV-protein marker enriched samples. Comparisons between PCa groups revealed mostly unique differentially expressed miRNAs. However, they targeted comprehensive and largely overlapping sets of cancer and progression-associated signalling, resistance, hormonal and immune pathways. Quantitative PCR confirmed changes in miR-892a (Gleason Score 7 vs. ≥8), miR-223-3p (progression vs. no progression) and miR-146a-5p (both comparisons). Their target mRNAs were expressed widely in PCa EV.CONCLUSIONS:PCa status and progression-linked RNAs in uEV are worth exploration in large personalized medicine trials.

Introduction: The bromodomain and extraterminal (BET) family of proteins are chromatin readers that promote the transcription of several important cell identity genes. BET proteins also control expression of many genes that play an essential role in the pathogenesis of human cancer, including cell-cycle and proliferation-regulating genes. The small-molecule BET inhibitors block BET binding to chromatin and have shown antitumor activity in a variety of pre-clinical cancer models. In this study, we evaluated the anticancer activity of the novel BET inhibitor, ODM-207, in ER+ breast cancer models as a single agent and in combination with other cancer drugs.Methods: ER+ breast cancer cell lines were studied for sensitivity to ODM-207 and the in vivo efficacy was assessed using the ER+ Ma3366 patient-derived xenograft model. For gene expression analyses, breast cancer cells were treated with ODM-207 or reference BET inhibitor JQ1 and differentially expressed genes were analyzed by RNA-sequencing. The ability of ODM-207 to regulate anticancer signaling pathways was validated by western blotting. Synergistic drug interactions were profiled using five-concentration dose response matrices.Results: ODM-207 is a novel BET inhibitor structurally distinct from JQ1 that shows antiproliferative activity in a broad panel of cancer cell lines. The strongest antitumor activity could be observed in hormone-dependent prostate and breast cancer models. In this study, we show that ODM-207 effectively inhibits the proliferation of ER+ breast cancer cell lines by inducing cell cycle arrest in G0/G1-phase. Additionally, ODM-207 suppresses the growth of ER+ patient-derived breast cancer xenograft tumors. ODM-207 as well as JQ1 targeted several pathways important for cancer progression such as MYC, estrogen response and cell cycle gene signatures. The inhibition of key cell cycle regulators, such as CDK4 and Cyclin D1, were further verified. The cyclin D1:CDK4/6 axis plays a significant role in the development, and currently, treatment of ER+ breast cancer together with endocrine therapy. Interestingly, ODM-207 was shown to synergize with palbociclib in vitro in ER+ breast cancer cell lines: the combination of ODM-207 and CDK4/6 inhibitor palbociclib achieved greater cell proliferation inhibition than either drug alone at sub IC50 concentrations. Notably, the ODM-207 and palbociclib combination did not cause the induction of an obvious senescent-like phenotype as compared to palbociclib alone, but rather affected cell survival cellular assays.Conclusions: In summary, ODM-207, which is currently in Phase I clinical trials for treating solid tumors, causes significant growth inhibition in pre-clinical models of ER+ breast cancer and enhances antiproliferative activity of palbociclib, providing a rationale for development of a combination therapy.Citation Format: Julia Lindqvist, Mari Björkman, Reetta Riikonen, Daniel Nicorici, Elina Mattila, Mahaboobi Jaleel, Chandrasekhar Abbineni, Anu-Maarit Moilanen. Antitumor activity of ODM-207, a novel BET bromodomain inhibitor, in nonclinical models of ER+ breast cancer as single agent and as a combination treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3827.

The role of adrenal androgens as drivers for castration-resistant prostate cancer (CRPC) growth in humans is generally accepted; however, the value of preclinical mouse models of CRPC is debatable, because mouse adrenals do not produce steroids activating the androgen receptor. In this study, we confirmed the expression of enzymes essential for de novo synthesis of androgens in mouse adrenals, with high intratissue concentration of progesterone (P-4) and moderate levels of androgens, such as androstenedione, testosterone, and dihydrotestosterone, in the adrenal glands of both intact and orchectomized (ORX) mice. ORX alone had no effect on serum P-4 concentration, whereas orchectomized and adrenalectomized (ORX + ADX) resulted in a significant decrease in serum P-4 and in a further reduction in the Low levels of serum androgens (androstenedione, testosterone, and dihydrotestosterone), measured by mass spectrometry. In line with this, the serum prostate-specific antigen and growth of VCaP xenografts in mice after ORX + ADX were markedly reduced compared with ORX alone, and the growth difference was not abolished by a glucocorticoid treatment. Moreover, ORX + ADX altered the androgen-dependent gene expression in the tumors, similar to that recently shown for the enzalutamide treatment. These data indicate that in contrast to the current view, and similar to humans, mouse adrenals synthesize significant amounts of steroids that contribute to the androgen receptor dependent growth of CRPC.

Abstract Introduction: The bromodomain and extraterminal (BET) family of proteins are chromatin readers that recognize and bind to specific acetylated histones and promote the transcription of several important cell identity genes. BET bromodomain inhibitors have shown promising antitumor activity in a variety of pre-clinical cancer models, as BET inhibition abrogates the transcription of several key oncogenes in a cell type-specific manner. It is known that inhibition of BET proteins effectively inhibits the proliferation of estrogen receptor positive (ER+) breast cancer cells, at least in part through repression of ER and MYC signaling. However, many additional cancer-associated genes are likely to underlie the growth inhibitory effects of BET inhibitors in breast cancer. The purpose of this study was to determine the anticancer activity of the novel BET bromodomain inhibitor ODM-207 in pre-clinical ER+ breast cancer models, and further, to look for cancer-associated signaling pathways suppressed by BET inhibitors. Methodology and results: ODM-207 is a novel, highly selective BET bromodomain inhibitor structurally distinct from JQ1 and its benzodiazepine-related derivatives. In this study, we show that ODM-207 effectively inhibits the proliferation of ER+ breast cancer cell lines when measured by cell viability assays as well as suppresses the growth of patient-derived xenograft tumors. Furthermore, we wanted to investigate the anticancer signaling pathways regulated by ODM-207 as well as the prototypical BET inhibitor JQ1 in breast cancer cells. For this purpose, we performed RNA sequencing on two ER+ breast cancer cell lines after 24h treatment with the aforementioned BET inhibitors. We found that both BET inhibitors targeted several genes and pathways important for breast cancer progression. For example, the targets included CDK4 and CDK6, two cell cycle kinases fundamental for the development and treatment of ER+ breast cancer. The RNA sequencing results were further validated in vitro, and were utilized as a basis for combination therapy assessment. Conclusions: Our results indicate that the novel BET bromodomain inhibitor ODM-207, which is currently in Phase I clinical trials for treating solid tumors, causes significant growth inhibition and cell cycle arrest in pre-clinical models of ER+ breast cancer, and regulates multiple crucial signaling pathways involved in breast cancer cell cycle and survival. Citation Format: Julia Lindqvist, Mari Björkman, Reetta Riikonen, Daniel Nicorici, Elina Mattila, Chandrasekhar Abbineni, Mahaboobi Jaleel, John Eriksson, Pekka Kallio, Anu-Maarit Moilanen. Therapeutic targeting of estrogen receptor positive breast cancer with the BET bromodomain inhibitor ODM-207 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3970.

Background: The bromodomain and extraterminal (BET) family of proteins are chromatin readers that promote the transcription of several important cell identity genes. BET inhibitors have shown promising antitumor activity in a variety of pre-clinical cancer models, as BET inhibition abrogates the transcription of several key oncogenes in a cell type-specific manner. Hence, the purpose of this study was to determine the anticancer activity of the novel BET inhibitor ODM-207 in ER+ breast cancer models and to look for cancer-associated signaling pathways suppressed by BET inhibitors. Methods: ER+ breast cancer cell lines were studied for sensitivity to ODM-207 and the in vivo efficacy was assessed using the ER+ Ma3366 patient-derived xenograft model. For gene expression analyses, breast cancer cells were treated with ODM-207 or reference BET inhibitor JQ1 and differentially expressed genes were analysed by RNA-sequencing. The ability of ODM-207 to regulate anticancer signaling pathways was validated by western blotting. Synergistic drug interactions were profiled using five-concentration dose response matrices. Results: ODM-207 is a novel BET inhibitor structurally distinct from JQ1 and its benzodiazepine-related derivatives. In this study, we show that ODM-207 effectively inhibits the proliferation of ER+ breast cancer cell lines as well as suppresses the growth of patient-derived xenograft tumors. Furthermore, ODM-207 and the JQ1 targeted several pathways important for cancer progression such as the DNA damage and repair pathways. Conclusions: Our results indicate that ODM-207, which is currently in Phase I clinical trials for treating solid tumors, causes significant growth inhibition in pre-clinical models of ER+ breast cancer, and regulates signaling pathways involved in breast cancer cell survival. Legal entity responsible for the study: Orion Corporation, Orion Pharma. Funding: Orion Corporation, Orion Pharma. Disclosure: A. Moilanen, M. Björkman, R. Riikonen, D. Nicorici, E. Mattila, P. Kallio: Employee: Orion Corporation, Orion Pharma. J. Lindqvist: Employee: Orion Corporation, Orion Pharma, Åbo Akademi University. C. Abbineni, M. Jaleel: Employee: Aurigene Discovery Technologies Limited. J. Eriksson: Employee: Åbo Akademi.

FusionCatcher is a software tool for finding somatic fusion genes in paired-end RNA-sequencing data from human or other vertebrates. FusionCatcher achieves competitive detection rates and real-time PCR validation rates in RNA-sequencing data from tumor cells. FusionCatcher is available at http://code.google.com/p/fusioncatcher