Oncology
Abstract
Mutations that initiate AML can cause clonal expansion without transformation (“clonal hematopoiesis”). Cooperating mutations, usually in signaling genes, are needed to cause overt disease, but these may require a specific “fitness state” to be tolerated. Here, we show that nearly all AMLs arising in a mouse model expressing two common AML initiating mutations (Dnmt3aR878H and Npm1cA) acquire a single copy amplification of chromosome 7, followed by activating mutations in signaling genes. We show that overexpression of a single gene on chromosome 7 (Gab2, which coordinates signaling pathways) is tolerated in the presence of the Npm1cA mutation, can accelerate the development of AML, and is important for survival of fully transformed AML cells. GAB2 is likewise overexpressed in many human AMLs with mutations in NPM1 and/or signaling genes, and also in Acute Promyelocytic Leukemia initiated by PML::RARA; the PML::RARA fusion protein may activate GAB2 by directly binding to its 5′ flanking region. A similar pattern of GAB2 overexpression preceding mutations in signaling genes has been described in other human malignancies. GAB2 overexpression may represent an oncogene-driven adaptation that facilitates the action of signaling mutations, suggesting an important (and potentially targetable) “missing link” between the initiating and progression mutations associated with AML.
Authors
Michael H. Kramer, Stephanie N. Richardson, Yang Li, Tiankai Yin, Nichole M. Helton, Daniel R. George, Michelle Cai, Sai Mukund Ramakrishnan, Casey D.S. Katerndahl, Christopher A. Miller, Timothy J. Ley
Abstract
BRD4 is an epigenetic reader protein that regulates oncogenes such as myc in cancer. However, its additional role in shaping immune responses via regulation of inflammatory and myeloid cell responses is not yet fully understood. This work further characterized the multifaceted role of BRD4 in anti-tumor immunity. NanoString gene expression analysis of EMT6 tumors treated with a BRD4 inhibitor identified a reduction in myeloid gene expression signatures. Additionally, BRD4 inhibition significantly reduced myeloid derived suppressor cells (MDSC) in the spleens and tumors of mice in multiple tumor models and also decreased the release of tumor-derived MDSC growth and chemotactic factors. Pharmacologic inhibition of BRD4 in MDSC induced apoptosis and modulated expression of apoptosis regulatory proteins. A BRD4-myeloid specific knockout model suggested that the dominant mechanism of MDSC reduction after BRD4 inhibition was primarily through a direct effect on MDSC. BRD4 inhibition enhanced anti-PD-L1 therapy in the EMT6, 4T1, and LLC tumor models, and the efficacy of the combination treatment was dependent on CD8+ T cells and on BRD4 expression in the myeloid compartment. These results identify BRD4 as a regulator of MDSC survival and provide evidence to further investigate BRD4 inhibitors in combination with immune based therapies.
Authors
Himanshu Savardekar, Andrew Stiff, Alvin Liu, Robert Wesolowski, Emily Schwarz, Ian C. Garbarine, Megan C. Duggan, Sara Zelinskas, Jianying Li, Gabriella Lapurga, Alexander Abreo, Lohith Savardekar, Ryan Parker, Julia Sabella, Mallory J. DiVincenzo, Brooke Benner, Steven H. Sun, Dionisia Quiroga, Luke Scarberry, Gang Xin, Anup Dey, Keiko Ozato, Lianbo Yu, Merve Hasanov, Debasish Sundi, Richard C. Wu, Kari L. Kendra, William E. Carson III
Abstract
Gasdermin (GSDM) family proteins mediate tumor pyroptosis and impact cancer progression, but other than that, their involvement in the tumor immune microenvironment remains largely unknown. Here, we show that activation of GSDMD in human tumor specimens mainly occurs in tumor-infiltrating leukocytes. Significantly, GSDMD deficiency or its inactivation in CD4+ T cells disabled CD8+ T cell–mediated antitumor immunity and caused tumor outgrowth in mice. Further study uncovered that, via inducing IL-2 production, GSDMD was required for CD4+ T cells to provide help to CD8+ T cell function. Mechanistically, GSDMD was cleaved by TCR stimulation–activated caspase-8 to form GSDMD-N pores, which enhanced Ca2+ influx for IL-2 induction. Moreover, GSDMD activation and function were conserved in human CD4+ T cells and associated with favorable prognosis and improved response to anti–PD-1 immunotherapy in colonic and pancreatic cancer. We believe this study identifies a new nonpyroptotic role of GSDMD in tumor immunity, proposing GSDMD as a potential target for cancer immunotherapy.
Authors
Yihan Yao, Lingling Wang, Weiqin Jiang, Ning Wang, Mengjie Li, Wenlong Lin, Ting Zhang, Wanqiang Sheng, Xiaojian Wang
Abstract
Mutant KRAS has been implicated in driving a quarter of all cancer types. Although inhibition of the KRASG12C mutant protein has shown clinical promise, there is still a need for therapies that overcome resistance and target non-KRASG12C mutations. KRAS activates downstream MYC, which is also a challenging-to-drug oncoprotein. We have developed an “inverted” RNAi molecule with the passenger strand of a MYC-targeting siRNA fused to the guide strand of a KRAS-targeting siRNA. The chimeric molecule simultaneously inhibits KRAS and MYC, showing marked improvements in efficacy beyond the individual siRNA components. This effect is mediated by 5’-dT overhangs following endosomal metabolism. The synergistic RNAi activity led to a >10-40-fold improvement in inhibiting cancer viability in vitro. When conjugated to an epidermal growth factor receptor (EGFR)-targeting ligand, the chimeric siRNA was delivered to and internalized by tumor cells. As compared with individual targeting siRNAs, the chimeric design resulted in considerably improved metabolic stability in tumors, enhanced silencing of both oncogenes, and reduced tumor progression in multiple cancer models. This inverted chimeric design establishes proof-of-concept for ligand-directed, dual-silencing of KRAS and MYC in cancer and constitutes an innovative molecular strategy for co-targeting any two genes of interest, which has broad implications.
Authors
Yogitha S Chareddy, Hayden P. Huggins, Snehasudha S Sahoo, Lyla Stanland, Christina Gutierrez-Ford, Kristina M. Whately, Lincy Edatt, Salma H Azam, Matthew C. Fleming, Jonah Im, Alessandro Porrello, Imani Simmons, Jillian L. Perry, Albert A. Bowers, Martin Egli, Chad V. Pecot
Abstract
Authors
Alyssa M. Duffy, Anshika Goenka, Maryam I. Azeem, Azmain Taz, Sayalee V. Potdar, Sara A. Scott, Ellen Marin, Jonathan L. Kaufman, Craig C. Hofmeister, Nisha S. Joseph, Vikas A. Gupta, Sagar Lonial, Ajay K. Nooka, Madhav V. Dhodapkar, Kavita M. Dhodapkar
Elevated NR2F1 underlies the persistence of invasive disease after treatment of BRAF-mutant melanoma
Abstract
Despite the clinical success of targeted inhibitors in cutaneous melanoma, therapeutic responses are transient and influenced by the aged tumor microenvironment, and drug-tolerant residual cells seed resistance. Given the similarities between drug tolerance and cellular dormancy, we studied the dormancy marker, nuclear receptor subfamily 2 group F member 1 (NR2F1), in response to targeted therapy. We utilized BRAF-V600E inhibitors (BRAFi) plus MEK inhibitors (MEKi) in BRAF-mutant melanoma models since melanoma patients treated with this combination display minimal residual disease, but ultimately tumors relapse. Transcriptomic analysis of melanoma samples from patients treated up to 20 days with BRAFi + MEKi showed increased expression of NR2F1. Similarly, NR2F1 was highly expressed in the drug-tolerant invasive cell state of minimal residual disease in patient-derived and mouse-derived xenograft tumors on BRAFi + MEKi treatment. Overexpression of NR2F1 alone was sufficient to reduce BRAFi + MEKi effects on tumor growth in vivo as well as on cell proliferation, death, and invasion in vitro. NR2F1-overexpressing cells were enriched for hallmarks gene sets for mTORC1 signaling, and NR2F1 bound to the promoter regions of genes involved in mTORC1 signaling. These cells were sensitive to the combination of BRAFi, MEKi plus rapamycin in vitro and in vivo. Melanomas from aged mice, which are known to exhibit a decreased response to BRAFi + MEKi, displayed higher levels of NR2F1 compared to tumors from young mice. Depleting NR2F1 levels in an aged mouse melanoma model improved the response to targeted therapy. These findings show high NR2F1 expression in ‘invasive-state’ residual cells and that targeting NR2F1-high cells with mTORC1 inhibitors could improve outcomes in melanoma patients.
Authors
Manoela Tiago, Timothy J. Purwin, Casey D. Stefanski, Renaira Silva, Mitchell E. Fane, Yash Chhabra, Jelan I. Haj, Jessica L.F. Teh, Rama Kadamb, Weijia Cai, Sheera R. Rosenbaum, Vivian Chua, Nir Hacohen, Michael A. Davies, Jessie Villanueva, Inna Chervoneva, Ashani T. Weeraratna, Dan A. Erkes, Claudia Capparelli, Julio A. Aguirre-Ghiso, Andrew E. Aplin
Abstract
BACKGROUND. Endocrine therapy (ET) with tamoxifen (TAM) or aromatase inhibitors (AI) is highly effective against hormone receptor (HR) positive early breast cancer (BC), but resistance remains a major challenge. The primary objectives of our study were to understand the underlying mechanisms of primary resistance and to identify potential biomarkers. METHODS. We selected >800 patients in three sub-cohorts (Discovery, N=364, matched pairs), Validation 1, N=270, Validation 2, N= 176) of the West German Study Group (WSG) Adjuvant Dynamic marker-Adjusted Personalized Therapy (ADAPT) trial who underwent short-term pre-operative TAM or AI treatment. Treatment response was assessed by immunohistochemical labeling of proliferating cells with Ki67 before and after ET. We performed comprehensive molecular profiling, including targeted next-generation sequencing (NGS) and DNA methylation analysis using EPIC arrays, on post-treatment tumor samples. RESULTS.TP53 mutations were strongly associated with primary resistance to both TAM and AI. In addition, we identified distinct DNA methylation patterns in resistant tumors, suggesting alterations in key signaling pathways and tumor microenvironment composition. Based on these findings and patient age, we developed the Predictive Endocrine ResistanCe Index (PERCI). PERCI accurately stratified responders and non-responders in both treatment groups in all three sub-cohorts and predicted progression-free survival in an external validation cohort and in the combined sub-cohorts. CONCLUSION. Our results highlight the potential of PERCI to guide personalized endocrine therapy and improve patient outcomes. TRIAL REGISTRATION. WSG-ADAPT, ClinicalTrials.gov NCT01779206, Registered 2013-01-25, retrospectively registered.
Authors
Guokun Zhang, Vindi Jurinovic, Stephan Bartels, Matthias Christgen, Henriette Christgen, Leonie Donata Kandt, Lidiya Mishieva, Hua Ni, Mieke Raap, Janin Klein, Anna-Lena Katzke, Winfried Hofmann, Doris Steinemann, Ronald E. Kates, Oleg Gluz, Monika Graeser, Sherko Kuemmel, Ulrike Nitz, Christoph Plass, Ulrich Lehmann, Christine zu Eulenburg, Ulrich Mansmann, Clarissa Gerhauser, Nadia Harbeck, Hans H. Kreipe
Abstract
Inactivation of cyclin-dependent kinase 12 (CDK12) defines an immunogenic molecular subtype of prostate cancer characterized by genomic instability and increased intratumoral T cell infiltration. This study reveals that genetic or pharmacologic inactivation of CDK12 and its paralog CDK13 robustly activates stimulator of interferon genes (STING) signaling across multiple cancer types. Clinical cohort analysis shows that reduced CDK12/13 expression correlates with improved survival and response to immune checkpoint blockade (ICB). Mechanistically, CDK12/13 depletion or targeted degradation induces cytosolic nucleic acid release, triggering STING pathway activation. CDK12/13 degradation delays tumor growth and synergizes with anti-PD1 therapy in syngeneic tumor models, enhancing STING activity and promoting CD8+ T cell infiltration and activation within tumors. Notably, the anti-tumor effects of this combination require STING signaling and functional CD8+ T cells. These findings establish STING activation as the key driver of T cell infiltration and the immune-hot tumor microenvironment in CDK12 mutant cancers, suggesting that dual CDK12/13 inhibitors and degraders activate anti-tumor immunity and potentiate responses to immunotherapies.
Authors
Yi Bao, Yu Chang, Jean Tien, Gabriel Cruz, Fan Yang, Rahul Mannan, Somnath Mahapatra, Radha Paturu, Xuhong Cao, Fengyun Su, Rui Wang, Yuping Zhang, Mahnoor Gondal, Jae Eun Choi, Jonathan K. Gurkan, Stephanie J. Miner, Dan R. Robinson, Yi-Mi Wu, Licheng Zhou, Zhen Wang, Ilona Kryczek, Xiaoju Wang, Marcin Cieslik, Yuanyuan Qiao, Alexander Tsodikov, Weiping Zou, Ke Ding, Arul M. Chinnaiyan
Abstract
The germinal center (GC) dark zone (DZ) and light zone (LZ) represent distinct anatomical regions in lymphoid tissue where B-cell proliferation, immunoglobulin diversification, and selection are coordinated. Diffuse Large B-cell Lymphomas (DLBCL) with DZ-like gene expression profiles exhibit poor outcomes, though reasons are unclear and are not directly related to proliferation. Physiological DZs exhibit an exclusion of T-cells, prompting exploration for whether T-cell paucity contributes to DZ-like DLBCL. We used spatial transcriptomic approaches to achieve higher resolution of T-cell spatial heterogeneity in the GC and to derive potential pathways that underlie T-cell exclusion. We showed that T-cell exclusion from the DZ was linked to DNA damage response (DDR) and chromatin compaction molecular features characterizing the spatial DZ signature, and that these programs were independent of AID deaminase activity. As ATR is a key regulator of DDR, we tested its role in the T-cell inhibitory DZ transcriptional imprint. ATR inhibition reversed not only the DZ transcriptional signature but also DZ T-cell exclusion in DZ-like DLBCL in vitro microfluidic models and in in vivo samples of murine lymphoid tissue. These findings highlight that ATR activity underpins a physiological scenario of immune silencing. ATR inhibition may reverse the immune silent state and enhance T-cell based immunotherapy in aggressive lymphomas with GC DZ-like characteristics.
Authors
Valeria Cancila, Giorgio Bertolazzi, Allison S.Y. Chan, Giovanni Medico, Giulia Bastianello, Gaia Morello, Daniel Paysan, Clemence Lai, Liang Hong, Girija Shenoy, Patrick W. Jaynes, Giovanna Schiavoni, Fabrizio Mattei, Silvia Piconese, Maria V. Revuelta, Francesco Noto, Luca Businaro, Adele De Ninno, Ilenia Cammarata, Fabio Pagni, Saradha Venkatachalapathy, Sabina Sangaletti, Arianna Di Napoli, Giada Cicio, Davide Vacca, Silvia Lonardi, Luisa Lorenzi, Andrés J.M. Ferreri, Beatrice Belmonte, Min Liu, Manikandan Lakshmanan, Michelle S.N. Ong, Biyan Zhang, Tingyi See, Kong-Peng Lam, Gabriele Varano, Mario P. Colombo, Silvio Bicciato, Giorgio Inghirami, Leandro Cerchietti, Maurilio Ponzoni, Roberta Zappasodi, Evelyn Metzger, Joseph Beechem, Fabio Facchetti, Marco Foiani, Stefano Casola, Anand D. Jeyasekharan, Claudio Tripodo
Abstract
Thiopurines are anticancer agents used for the treatment of leukemia and autoimmune diseases. These purine analogs are characterized by a narrow therapeutic index because of the risk of myelosuppression. With the discovery of NUDIX hydrolase 15 (NUDT15) as a major modulator of thiopurine metabolism and toxicity, we sought to comprehensively examine all members of the NUDIX hydrolase family for their effect on the pharmacologic effects of thiopurine. By performing a NUDIX-targeted CRISPR/Cas9 screen in leukemia cells, we identified NUDT5, whose depletion led to drastic thiopurine resistance. NUDT5 deficiency resulted in a nearly complete depletion of active metabolites of thiopurine and the loss of thioguanine incorporation into DNA. Mechanistically, NUDT5 deletion resulted in substantial alteration in purine nucleotide biosynthesis, as determined by steady-state metabolomics profiling. Stable isotope tracing demonstrated that the loss of NUDT5 was linked to a marked suppression of the purine salvage pathway but with minimal effects on purine de novo synthesis. Finally, we comprehensively identified germline genetic variants in NUDT5 associated with thiopurine-induced myelosuppression in 582 children with acute lymphoblastic leukemia. Collectively, these results pointed to NUDT5 as a key regulator of the thiopurine response primarily through its effects on purine homeostasis, highlighting its potential to inform individualized thiopurine therapy.
Authors
Maud Maillard, Rina Nishii, Hieu S. Vu, Kashi R. Bhattarai, Wenjian Yang, Jing Li, Ute Hofmann, Daniel Savic, Smita Bhatia, Matthias Schwab, Min Ni, Jun J. Yang
Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)