MYC Activity Research Articles

TDP2 is a regulator of estrogen-responsive oncogene expression.

With its ligand estrogen, the estrogen receptor (ER) initiates a global transcriptional program, promoting cell growth. This process involves topoisomerase 2 (TOP2), a key protein in resolving topological issues during transcription by cleaving a DNA duplex, passing another duplex through the break, and repairing the break. Recent studies revealed the involvement of various DNA repair proteins in the repair of TOP2-induced breaks, suggesting potential alternative repair pathways in cases where TOP2 is halted after cleavage. However, the contribution of these proteins in ER-induced transcriptional regulation remains unclear. We investigated the role of tyrosyl-DNA phosphodiesterase 2 (TDP2), an enzyme for the removal of halted TOP2 from the DNA ends, in the estrogen-induced transcriptome using both targeted and global transcription analyses. MYC activation by estrogen, a TOP2-dependent and transient event, became prolonged in the absence of TDP2 in both TDP2-deficient cells and mice. Bulk and single-cell RNA-seq analyses defined MYC and CCND1 as oncogenes whose estrogen response is tightly regulated by TDP2. These results suggest that TDP2 may inherently participate in the repair of estrogen-induced breaks at specific genomic loci, exerting precise control over oncogenic gene expression.

Abstract LB295: Unsaturated fatty acid synthesis is associated with poor prognosis and is differentially regulated by MYCN and tumor suppressor microRNAs in neuroblastoma

Abstract Dysregulated metabolism is a hallmark of cancer. While the role of glucose metabolism in cancer has been extensively studied, relatively little has been done to understand the role of poly-unsaturated fatty acids in cancer biology. To gain insight into their contribution to cancer progression we determined the expression patterns and outcome associations of both saturated and unsaturated fatty acid synthesis (UFAS) genes in pediatric neuroblastoma (NB), and fatty acid levels in NB cell lines derived from high-risk tumors. MYCN amplification and disruption of tumor suppressor miRNAs (TSmiR) function are central drivers of poor outcomes in NB. MYC, MYCN, and TSmiRs regulate glucose metabolism; however, their role in UFAS remains poorly understood. Using complementary analysis of publicly available genomic datasets and cultured NB cell measurements we examined the potential regulation of UFAS genes through MYCN and MYC activity and posttranscriptional regulation by TSmiRs. Here we show that de novo and UFAS pathway genes FASN, ELOVL6, SCD, FADS2, and FADS1 are upregulated in high-risk NB and are associated with poor prognosis. RNA-Seq analysis of eight human NB cell lines revealed parallel UFAS gene expression patterns. Consistent with this, we found that NB-related TSmiRs were predicted to extensively target these genes. Additionally, we observed that both MYC and MYCN upregulated UFAS pathway genes while suppressing TSmiR host gene expression, thereby creating a possible UFAS regulatory network between MYCN and TSmiRs in NB. Furthermore, using mass spectrometry analysis we found NB cells to be high in de novo synthesized omega 9 (ω9) unsaturated fatty acids and low in both ω6 and ω3. This finding provides a plausible means for NB to limit cell-autonomous immune stimulation and reactive oxygen species (ROS)-driven apoptosis from ω6 and ω3 unsaturated fatty acid derivatives, respectively. We propose a model in which the UFAS pathway, through novel regulation by MYCN and TSmiRs, plays a key role in neuroblastoma pathology with implications for other MYC-driven cancers. Citation Format: Dennis A. Sheeter, Secilia Garza, Hui Gyu Park, Lorraine-Rana Benhamou, Nikki R. Badi, Erika C. Espinosa, Kumar Kothapalli, J. Thomas Brenna, John T. Powers. Unsaturated fatty acid synthesis is associated with poor prognosis and is differentially regulated by MYCN and tumor suppressor microRNAs in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB295.

Abstract LB005: Identification of novel targets for MYC-driven hepatocellular carcinoma via transomics

Abstract Hepatocellular carcinoma (HCC) is an aggressive tumor, and treatment options for patients with unresectable HCC are limited to targeted therapies and immunotherapy. The activation and dysregulation of MYC have been implicated in multiple cancers, including HCC, where approximately 30% of human HCC samples show MYC gene amplification. However, it is challenging to target MYC directly. Therefore, we applied a novel approach to circumvent targeting MYC directly by leveraging Pepper’s proprietary transomic analysis platform, COMPASS, to identify novel targets that are predicted to mimic the effect of turning MYC off. Specifically, COMPASS unlocks functional drivers of disease to identify novel drug targets. To study the role of MYC in tumor biology, we utilize an HCC cell line with tunable MYC expression. We generate genomic, transcriptomic, proteomic, and phosphoproteomic data from samples with MYC-on and MYC-off and compare via COMPASS to identify novel targets that mimic turning MYC off. The MYC-conditional HCC cell line (EC4) allows the regulation of MYC expression via the tetracycline regulatory (Tet-Off) system. Four omic datasets were collected from each sample: genomics (next-generation sequencing, NGS), transcriptomics (NGS), proteomics (mass spectrometry), and phosphoproteomics (mass spectrometry). We employed the COMPASS target prioritization algorithm to identify and rank novel targets that mimic turning MYC off. Targets were then filtered to select those with an available pharmacological tool compound (PTC). The PTCs were used to evaluate the targets in the Huh-7 xenograft model of HCC. All targets were kinases or related to kinase activity and pathways. All PTCs inhibited tumor growth from 18.1-78.3%. One multi-tyrosine kinase inhibitor resulted in maximum tumor growth inhibition, resulting in stasis of tumor growth and increased survival compared with control vehicle-treated mice. We have previously shown how the COMPASS transomics analysis approach can identify novel drug targets for drug resistant EGFRm NSCLC. The data presented here on MYC-driven HCC further validates this approach for novel target identification. Ongoing work will investigate novel targets using gene silencing, as PTCs were unavailable for many of these novel high-ranked targets identified in this study. Further validation studies are ongoing against other difficult-to-treat MYC-driven cancers to identify novel clinical targets and drug candidates to circumvent the MYC pathway. Citation Format: Simon P. Fricker, Christopher J. Nicholson, Samuel J. Roth, Arudhir Singh, Caitlin Brown, Jon Hu, Petronela Buiga, Vishnu P. Kanakaveti, Anja Deutzmann, Dean Felsher, Samantha D. Strasser. Identification of novel targets for MYC-driven hepatocellular carcinoma via transomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB005.

Abstract CT158: A dose escalation and cohort expansion study of the CDK9 inhibitor KB-0742 in relapsed, refractory and transcriptionally addicted solid tumors

Abstract Background MYC transcriptional deregulation is a hallmark of cancer and is associated with poor clinical outcomes. MYC deregulated solid tumors including non-small cell (NSCLC), small cell lung cancer (SCLC), triple negative breast cancer (TNBC), and ovarian cancer are known for their aggressiveness and frequent relapse. Analysis of a real-world solid tumor cohort including NSCLC (n= 20,470), SCLC (n=1,517), TNBC (n=2,576) and ovarian cancer (n=1,667) demonstrated MYC family overexpression and/or genomic amplification in 47% to 87% of patients. Although MYC remains difficult to drug, targeting of its cofactors has emerged as an attractive strategy to inhibit MYC oncogenic activity. Cyclin-dependent kinase 9 (CDK9), a MYC cofactor, is a critical regulator of oncogenic MYC expression and activity. KB-0742 is a potent, selective, and orally bioavailable inhibitor of CDK9 with a long plasma half-life. KB-0742 is being evaluated in an ongoing Phase 1/2 study in advanced solid tumors (NCT04718675). Methods The Phase 1/2 study is being conducted in two parts: dose escalation and dose expansion. Part 1 dose escalation comprises patients with relapsed or refractory solid tumors. Part 2 dose expansion comprises patients with solid tumors that have a high prevalence of MYC overexpression including ovarian cancer, NSCLC, TNBC and SCLC. KB-0742 is dosed orally once daily for 3 consecutive days followed by 4 days off on a weekly basis in 28-day cycles until unacceptable toxicity or disease progression. Eligibility criteria include age > 18 years acceptable organ function and ECOG PS < 2. Up to 170 patients are planned to be enrolled in the study. Primary objectives include evaluation of pharmacokinetics (PK), pharmacodynamics (PD), safety, tolerability, and preliminary anti-tumor activity with the goal of identifying a maximum tolerated dose (MTD). Plasma PK measurements include Cmax, tmax, AUC0-last, accumulation ratio (Racc) and t1/2. Safety data will be evaluated per the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) v5.0. RP2D is informed by PD in peripheral blood mononuclear cells using assays to evaluate phosphorylation of the CDK9 substrate serine 2 on the RNA Polymerase II C-terminal domain (pSER2) and CDK9-responsive gene expression. Radiographic tumor response is assessed using RECIST 1.1 criteria. Exploratory objectives include assessment of KB-0742 PD in tumor tissue and profiling of treatment-related genomic, transcriptomic, and proteomic changes. The study is continuing as planned; the next data analysis will occur in 2024. Citation Format: Miguel Villalona Calero, Mark Agulnik, Monica Mita, Alain Mita, Noah Federman, Drew Rasco, David Spigel, Jia Luo, Glenn Hanna, Gregory Cote, Mohamad A. Salkeni, Rashmi Chugh, Natraj R. Ammakkanavar, Satish A. Shah, Amol Rao, Kamalesh Kumar Sankhala, Richard E. Cutler, Tressa Hood, Luis Carvajal, Crystal MacKenzie, Charles Lin, Jorge DiMartino, Elizabeth A. Olek, Brian Van Tine. A dose escalation and cohort expansion study of the CDK9 inhibitor KB-0742 in relapsed, refractory and transcriptionally addicted solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT158.

Identification of spatially-resolved markers of malignant transformation in Intraductal Papillary Mucinous Neoplasms

The existing Intraductal Papillary Mucinous Neoplasm (IPMN) risk stratification relies on clinical and histological factors, resulting in inaccuracies and leading to suboptimal treatment. This is due to the lack of appropriate molecular markers that can guide patients toward the best therapeutic options. Here, we assess and confirm subtype-specific markers for IPMN across two independent cohorts of patients using two Spatial Transcriptomics (ST) technologies. Specifically, we identify HOXB3 and ZNF117 as markers for Low-Grade Dysplasia, SPDEF and gastric neck cell markers in borderline cases, and NKX6-2 and gastric isthmus cell markers in High-Grade-Dysplasia Gastric IPMN, highlighting the role of TNFα and MYC activation in IPMN progression and the role of NKX6-2 in the specific Gastric IPMN progression. In conclusion, our work provides a step forward in understanding the gene expression landscapes of IPMN and the critical transcriptional networks related to PDAC progression.

Abstract 3043: MYC activation and PTEN loss synergistically activate proliferation in a mouse model of lethal prostate cancer

Abstract Prostate cancer kills 30,000 men per year in the U.S. Yet, of all primary prostate cancers, only a relatively small percentage are aggressive and have the potential to become life threatening. In terms of genomic copy number alterations, concurrent amplification of MYC and loss of tumor suppressor PTEN is the strongest predictor of prostate cancer specific mortality. We have previously shown, in a novel mouse model, that combined MYC overexpression and Pten loss results in the development of precursor lesions (PIN) that lead to aggressive metastatic prostatic adenocarcinoma, yet overexpression of MYC alone or Pten loss alone result only in precursor lesion development. Here, we performed genome wide transcriptome analysis using bulk RNA seq to obtain clues regarding the synergy between MYC overexpression and Pten loss. Prostates harboring PIN lesion were dissected from mice engineered to have MYC activation alone, Pten loss alone, and the combination thereof (BMPC mice), as well as invasive primary adenocarcinomas and metastatic lesions from BMPC mice. Principal Components Analysis (PCA) analysis separated the lesions into highly distinct groups. Genes contributing the most to the first principal component were related to inflammation and cell proliferation/cell cycle progression by functional annotation. The proliferation-related genes were increased somewhat by MYC overexpression alone, were not increased Pten loss, and were more consistently increased by combined MYC overexpression and Pten loss in BMPC. Strikingly, mice with combined MYC overexpression and Pten loss showed approximately 2-4 fold higher Ki67 proliferation index than mice with MYC activation alone. PIN lesions driven by MYC overexpression or PTEN loss alone were characterized by Gene Set Enrichment Analysis (GSEA). Among the pathways with the highest negative enrichment scores in PIN samples with PTEN loss were those that generally describe ribosome biogenesis and translation. PIN lesions with Pten loss showed lower hybridization signals for the 45S rRNA, which was increased by MYC alone and somewhat more in BMPC PIN lesions. Our results demonstrate that combined activation of MYC and Pten loss drive increased cell proliferation beyond that seen with either gene alteration alone. These findings suggest that a key part of the molecular mechanisms by which MYC and PTEN alterations may synergize to drive aggressive prostate cancer is related to increased tumor cell proliferation. Surprisingly, we uncovered the novel finding that Pten loss, in the absence of MYC overexpression, is associated with decreased expression of genes coding for proteins that mediate translation, as well as decreased 45S pre-rRNA. Since ribosome biogenesis is a key driver of cell proliferation, it is likely that loss of both copies of PTEN alone, in the absence of MYC overexpression, would be selected against in early lesions in prostate cancer. Citation Format: Michael Rubenstein, Tracy Jones, Jessica Hicks, Apurv Rege, Qizhi Zheng, Carolina Gomes-Alexandre, Gretchen Hubbard, Srinivasan Yegnasubramanian, Charles Bieberich, Angelo DeMarzo. MYC activation and PTEN loss synergistically activate proliferation in a mouse model of lethal prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3043.

Abstract 518: The CDK2/9 inhibitor fadraciclib is active in Richter transformation and lymphoma cell lines by targeting both cell survival and proliferation

Abstract Richter transformation (RT), characterized by the transformation of chronic lymphocytic leukemia (CLL) to an aggressive large-cell lymphoma, remains a challenge for the therapy. Genetic aberrations, such as Myc activation, TP53 abnormality, NOTCH1 gene mutation or loss of CDKN2A are associated with uncontrolled RT cell proliferation and escaping of apoptosis. Among those changes, gain of Myc activity, found in around 70% of RT, plays a nexus role in the pathogenesis of RT. As a transcription factor, Myc activation has a profound effect on cell survival, proliferation, adhesion and metabolism. Because of its essential role in lymphoma transformation and aggressiveness, Myc is a vital target for therapy toward RT. Fadraciclib (CYC065) is a second-generation CDK inhibitor with selective potency toward CDK2 and CDK9. We have shown previously that by inhibiting CDK9-mediated transcription, fadraciclib reduced expression of the short-lived anti-apoptotic protein Mcl-1 to initiate apoptosis in primary CLL cells. Like Mcl-1, Myc is a quintessential example of an oncogene with rapid turnover in both its mRNA and protein, therefore a promising target of CDK9 inhibition. Thus, we proposed that fadraciclib would target both the survival and proliferation pathways of RT pathogenesis through collaborative inhibition of Mcl-1, Myc and CDK2. First, our data showed that fadraciclib effectively induced apoptosis in the RT cell line HPRT1, as well as the lymphoma cell lines with Myc amplification, including Raji, Ramos and SU-DHL-2. The IC50 for apoptosis was 0.3 µM at 24h in the HPRT1 cells, which is less than the average IC50 for the primary CLL cells (0.8 µM). The IC50 for the lymphoma lines ranged from 0.8 µM to 1.3 µM. Immunoblots showed reduction of RNA Pol2 phosphorylation by fadraciclib, consistent with the inhibition of CDK9. As a result of loss of RNA Pol2 activity and pausing of transcription, there was a clear reduction of both Mcl-1 and Myc mRNA, measured by real-time RT-PCR, that was associated with the reduction of their protein levels. CDK2 was also inhibited by fadraciclib, shown by the reduction of the phosphorylation of both RB and NPM. Second, Fadraciclib inhibited cell proliferation of the HPRT1 cells at an IC50 of 0.25 µM measured by the CellTiter-Glo assay. The IC50 for growth inhibition in the lymphoma lines ranged from 0.5 to 0.7 µM. CFSE tracing demonstrated halting of cell division upon fadraciclib incubation. Cell cycle analysis by Click-iT EdU showed reduction of S-phase population and arresting of cells in both G1 and G2. We are currently working on dissecting the contributions of inhibiting Myc, Mcl-1 and CDK2 individually and together, in apoptosis induction and proliferation blockade. Taken together, these data suggest that fadraciclib is active in RT cells and Myc-dependent lymphoma cell lines and support expansion of its clinical testing in patients with RT. Citation Format: Rong Chen, Yuling Chen, Ping Xiong, William Plunkett. The CDK2/9 inhibitor fadraciclib is active in Richter transformation and lymphoma cell lines by targeting both cell survival and proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 518.

Abstract 515: Endocycling cancer cells undergo multiple S phases while repressing myc activity

Abstract Chemotherapy resistance remains a major challenge when treating patients with metastatic cancer. While intrinsic resistance to therapy has been well studied, phenotypic plasticity that drives entry and exit of resistant cell states is poorly understood. One of these plastic cell states used by cancer cells under chemotherapy treatment is characterized by the reprogramming of the mitotic cell cycle to enter a non-proliferative endocycle. Cells in this endocycling state decouple DNA synthesis from cell division by actively progressing through G1, S, and G2 phases of the cell cycle while repeatedly skipping mitosis. After a dormant period, cells can switch back to a mitotic cell cycle to repopulate a tumor. The underlying mechanisms that drive this cell cycle reprogramming remain unknown. C-myc is an oncogenic transcription factor that is dysregulated in ~70% of human cancers. C-myc is responsible for the activation of several cell cycle drivers, including cyclins and CDKs, and represses cell cycle inhibitors, such as CDK inhibitors. High levels of myc activity allow cells to move through the cell cycle quickly with a rapid doubling time. Documented endocycles in polyploid cells rely on high c-myc activity; however, our data shows that endocycling cancer cells have repressed levels of c-myc protein and transcriptional activity. Transcriptomic data shows that myc target genes are expressed at very low levels compared to the mitotic parental cells, and complementary data shows low protein levels. While myc downregulation is expected in non-proliferative cells, the compensatory mechanisms mediating the G1/S transition in the cancer endocycle remain unknown. Recent data has shown that myc suppression alone is not sufficient to induce a sustained endocycle at a population level, despite premature APCCdh1 activation during G2 in a subset of cells. We hypothesize that restoring myc activity to the levels of mitotic parental cells will drive endocycling cells to resume a proliferative mitotic cell cycle and produce mitotic progeny cells. Understanding the mechanisms that govern this transition is crucial to generate next-generation cancer therapeutics to disrupt endocycling cells, blocking acquired resistance and disease recurrence. Citation Format: Michael A. Loycano, Kenneth J. Pienta, Sarah R. Amend. Endocycling cancer cells undergo multiple S phases while repressing myc activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 515.

Abstract 1779: De novo purine pathway enzyme ATIC promotes tumor progression and modulates metabolic reprogramming in breast cancer

Abstract Breast cancer (BC), a complex and heterogeneous disease, poses a significant global health challenge with a pressing need for enhanced prognostic markers and therapeutic targets. This study focuses on 5-Aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), an enzyme essential in de novo purine biosynthesis and previously implicated in myeloma and hepatocellular carcinoma progression. Leveraging publicly available datasets, including TCGA, METABRIC, and GENT2, we identified elevated ATIC expression in BC tissues compared to normal tissues. Notably, patients with elevated ATIC expression displayed significantly diminished overall survival rates, underscoring ATIC's potential as a prognostic marker. Functional assays involving ATIC downregulation in BC cell lines revealed suppressed cell growth, migration, and increased sensitivity to chemotherapeutic drug, Doxorubicin, implicating ATIC in BC progression. Further, RNA-sequencing analysis of ATIC knockdown (KD) BC cell lines revealed a positive correlation between ATIC expression and the activation of MYC and E2F-regulated pathways. The marked attenuation in cell growth and various tumor properties upon ATIC KD provides mechanistic insights into the role of ATIC in fueling BC progression, potentially through the modulation of MYC and E2F target gene expression. Gene Set Enrichment Analysis (GSEA) on differentially expressed genes identified between tumors with high ATIC expression and low ATIC expression in BC patients from the TCGA cohort revealed a notable enrichment of genes associated with MYC and E2F targets, providing additional support for the observed molecular associations in our experimental model. Intriguingly, ATIC KD in Triple Negative BC cell line unexpectedly led to an increase in fatty acid β-oxidation (FAO), uncovering a metabolic vulnerability in these cells. In conclusion, this study unravels the multifaceted role of ATIC in BC, demonstrating its influence on BC tumor progression and metabolism. The observed increase in FAO upon ATIC KD presents a novel metabolic vulnerability that could be explored for targeted therapeutic interventions in BC. This comprehensive analysis contributes valuable insights toward understanding ATIC as a potential therapeutic target and prognostic indicator in BC. Citation Format: Divya Murthy, Kuldeep S. Attri, Jun Hyoung Park, Benny A. Kaipparettu. De novo purine pathway enzyme ATIC promotes tumor progression and modulates metabolic reprogramming in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1779.

Abstract 7353: Exploring intrapatient heterogeneity in metastatic breast cancer: Insights into tumor evolution from a warm procurement trial

Abstract Metastatic breast cancers show variable clinical responses due to inherent heterogeneity, both within tumors and among patients. Studies mainly examine molecular diversity across different patients' tumors and genetic variance within a single tumor. Yet, the variability among multiple tumors or metastases in a single patient remains underexplored. Our study investigates this intrapatient heterogeneity, examining cell and tumor evolution from a single genetic source in metastatic breast cancer. In a warm procurement trial involving 6 patients with multi-site tumors, samples were collected for detailed molecular analysis. Whole exome sequencing (WES) and single-cell RNA sequencing (scRNA-seq) were used to analyze genetic similarities and disparities. WES data revealed mutational patterns and copy number alterations, showing homogeneity and heterogeneity in tumors within patients. Further, phylogenetic analysis with scRNA-seq variant calls paired with transcriptional phenotypes uncovered a MYC-enriched subclonal population in a metastatic site. Integrating WES and scRNA-seq data provided a clearer understanding of the genomic and phenotypic features in and between patients. Our WES study revealed significant interpatient variations in genetic drivers and CNA profiles. Among five patients, two showed APOBEC and HR mutations, linked to increased tumor mutations. Despite general genetic homogeneity, there were exceptions. Tumors in Patient 3's upper lymph nodes had distinct genetics compared to abdominal tumors. Unique genetic patterns were also noted in the pancreas and mesentery of Patients 3 and 7, respectively. Genetic findings were supported by transcriptional analysis, showing distinct expression patterns in specific sites. Phylogenetic analysis highlighted intratumor heterogeneity, as shown by variations in phylogenetic composition and gene expression across samples. Notably, in patients with the most samples (Patients 3 and 7), a conserved cell clade, evolved early and primarily located in a single site, displayed a high proliferation gene expression signature closely associated with Myc activity. This underscores the importance of early clonal expansions in tumor evolution and suggests potential therapeutic targets in these early, proliferative cells. Our study highlights the vital role of intrapatient heterogeneity in understanding cancer complexity and treatment resistance. By analyzing genetic and phenotypic variability in multi-site tumors within patients, and Myc's key role in tumor growth and evolution, our research offers new insights into tumor evolution. This underscores the need for personalized oncology treatments based on each patient's tumor genetics. Citation Format: Isaac Bishara, Xuan Liu, Jason Griffiths, Jiayi Liu, Patrick Cosgrove, Jasmine R. McQuerry, Feng Chi, Pierre Wallet, Vince K. Grolmusz, Benjamin Copeland, Lance Pflieger, Jinfeng Chen, Sumana Majumdar, Grace Ronquillo, Terron Crowder, Rena Emond, Rachel Factor, Eliza Barragan, David Bowtell, Adam Cohen, Daniel D. Schmolze, Peter Lee, Kena Ihle, Lusine Tumyan, Aritro Nath, James Waisman, Andrea Bild, Jeff Chang. Exploring intrapatient heterogeneity in metastatic breast cancer: Insights into tumor evolution from a warm procurement trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7353.

Abstract 6944: Transcriptional programs underpinning reoccurring cancer cell states, plasticity and tumour phylogenies in clear cell kidney cancer

Abstract Intratumor heterogeneity (ITH) is a substrate for tumor evolution and confers amenability to stressors and therapies. Sources of ITH include subclonal mutations, microenvironmental factors, and plasticity whereby a cell has the ability to adopt different cell states. Clear cell kidney cancer (ccRCC) has a well-characterized genetic initiating event - VHL inactivation - resulting in activation of HIF-1 and HIF-2 transcription factors. Although VHL inactivation is consistently clonal in nature, ccRCC nonetheless exhibits extensive genetic and morphological ITH. This makes ccRCC an excellent model in which to study ITH. We performed single cell RNA-seq on multiregional samples from kidney tumors and macroscopically normal kidney tissue. We obtained 55 samples from 16 tumors and used the 10x Genomics platform to obtain gene expression data from ~150,000 single cells, including ~80,000 cancer and normal epithelial cells. We identified three cancer cell states, co-existing in all clear cell kidney cancer tumors analyzed and marked by different gene expression programs: 1) differentiated-like cells marked by proximal tubule cell genes characteristic of the cancer cell of origin; 2) epithelial-mesenchymal transition (EMT)-like cells and 3) injured-like cells. Differentiated markers and EMT markers were associated with good and poor prognosis, respectively, indicating that these cell subpopulations are clinically important. Notably, these cell states are also present in the normal proximal tubule although in different proportions to the tumor samples, which contain more cells in the EMT-like and injured-like states. This suggests that the core features of ITH are inherent to the cell of origin of the cancer, but that the cell state trajectories are dysregulated in the tumor. Furthermore, in tumors these states exist as a continuum, whereby transitional cells in between states appear to exist, suggesting that cancer cells are continually and dynamically transitioning between states. Lastly, we used chromosomal copy number variation (CNV) patterns to study genetic ITH. We examined the effect of 14q loss, which is associated with ccRCC metastasis, on gene expression. We found that 14q loss is associated with downregulation of HIF-1 transcriptional activity together with a reciprocal increase in HIF-2 activity, MYC activity and oxidative phosphorylation, highlighting potential mechanisms by which 14q loss may driver ccRCC metastasis. Citation Format: Olivia Lombardi, Ran Li, Lisa Browning, Faiz Jabbar, Hannah Evans, Peter J. Ratcliffe, David R. Mole. Transcriptional programs underpinning reoccurring cancer cell states, plasticity and tumour phylogenies in clear cell kidney cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6944.

Abstract 2004: CDC7 inhibition prevents neuroendocrine transformation in the lung and prostate through MYC degradation

Abstract Neuroendocrine (NE) transformation occurs as a mechanism of resistance to targeted therapy in up to 14% and 30% of EGFR-mutant lung and AR-dependent prostate adenocarcinomas, respectively, leading to poor prognosis. Even if we know the tumor population at high risk of transformation (TP53/RB1-mutated), no therapies to prevent NE relapse are currently available. To identify therapeutic vulnerabilities for tumors undergoing NE transformation, we performed an in vitro CRISPR screen in a NE-transformed lung tumor model. This screen identified CDC7, involved in DNA replication and DNA damage response, as a potential therapeutic target in this setting. Proteogenomic analyses revealed CDC7 upregulation in lung and prostate clinical samples undergoing NE transformation, detected already in pre-transformation adenocarcinomas. These results indicated that CDC7 expression is induced already at early steps of transformation. Consistently, TP53/RB1-mutated lung and prostate adenocarcinomas exhibited higher CDC7 expression than their double wild-type counterparts. Combined ChIP-seq and promoter reporter assays indicated that CDC7 expression was directly regulated by TP53 and RB1 inactivation. Importantly, TP53/RB1-inactivation induced sensitivity to the CDC7 inhibitor simurosertib, unraveling a therapeutic vulnerability in tumors at high risk of NE transformation. Thus, we tested the combination of simurosertib with targeted therapy in vivo in different lung and prostate patient-derived models of NE transformation, namely TP53/RB1-knock out adenocarcinomas known to undergo NE transformation on targeted therapy. In these, simurosertib was able to suppress NE transformation and dramatically delay tumor relapse. Trajectory analysis on single-cell transcriptomic data for such models revealed a NE transformation transcriptional program occurring already in the untreated tumors before transformation. Remarkably, simurosertib treatment reverted this transcriptomic state and induced a reversion to the original adenocarcinoma transcriptomic profile. CDC7 inhibition led to increased proteasomal activity and degradation of MYC, a stemness transcription factor involved in NE transformation. Ectopic overexpression of MYCT58A, a proteasome degradation-resistant MYC isoform, rescued the NE phenotype in these transformation models, suggesting that CDC7 inhibition-induced MYC degradation is the mechanism by which NE transformation is prevented. In sum, CDC7 inhibition may suppress, or at least dramatically delay NE transformation in patients with lung and prostate adenocarcinomas at high risk of transformation, by inducing MYC proteasomal degradation. The clinical availability of CDC7 inhibitors, currently in phase II clinical trials after demonstrating tolerability and preliminary efficacy, will allow rapid translation of these results into the clinics. Citation Format: Alvaro Quintanal-Villalonga, Fathema Uddin, Kenta Kawasaki, Esther Redin, Vidushi Durani, Amin Sabet, Wouter Karthaus, Yingqian A. Zhan, Samir Zaidi, Moniquetta Shaffer, Harsha Sridhar, Juan Qiu, Parvathy Manoj, Elisa De Stanchina, Michael C. Haffner, Charles L. Sawyers, Charles M. Rudin. CDC7 inhibition prevents neuroendocrine transformation in the lung and prostate through MYC degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2004.

Abstract 7072: MYC acetylated lysine residues drive oncogenic cell transformation and regulate select genetic programs for cell adhesion-independent growth and survival

Abstract The MYC oncogenic transcription factor is acetylated by the p300 and GCN5 histone acetyltransferases. The significance of MYC acetylation and functions of specific acetylated lysine (K) residues have remained unclear. Here, we show that the major p300-acetylated K148(149) and K157(158) sites in human (or mouse) MYC and the main GCN5-acetylated K323 residue are reversibly acetylated in various malignant and non-malignant cells. Oncogenic overexpression of MYC enhances its acetylation and alters regulation of site-specific acetylation by proteasome and deacetylase inhibitors. Acetylation of MYC at different K residues differentially affects its stability in a cell type- dependent manner. Lysine-to-arginine substitutions indicate that although none of the acetylated K (AcK) residues is required for MYC stimulation of adherent cell proliferation, individual AcK sites have gene-specific functions controlling select MYC-regulated processes in cell adhesion, contact inhibition, apoptosis, and/or metabolism, and are required for the malignant cell transformation activity of MYC. Each AcK site is required for anchorage-independent growth of MYC-overexpressing cells in vitro, and both the AcK148(149) and AcK157(158) residues are also important for the tumorigenic activity of MYC-transformed cells in vivo. These AcK residues may also preferentially activate transcription via facilitating MYC binding to several cancer-associated target gene promoters and via enhancing RNAP II recruitment. With the knowledge that MYC lysine residues can be acetylated by HATs GCN5 and P300, some HATs and HAT associated cofactors (i.e. YEATS2 and PIN1) are important for MYC recruitment to DNA binding. By identifying MYC acetylation-dependent coregulators and their oncogenic function, we proposed to understand whether and how MYC acetylation residues cooperate with its cofactors and influence malignant transformation in breast cancer cells.Overall, this study will uncover the functions and mechanisms of MYC acetylation in regulation of malignant transformation and the impact on breast cancer initiation and progression. By analyzing deregulated target genes and MYC-relevant coactivators, the MYC AcK site-specific signaling pathways identified may offer new avenues for selective therapeutic targeting of MYC oncogenic activities. Citation Format: Jeffrey J. Pino, Matthew Hurd, Yifan Zhao, Kay Jang, Michael Allevato, Marina Vorontchikhina, Wataru Ichikawa, Ryan Gates, Emily Villalpando, Michael Hamilton, Francesco Failo, Songqin Pan, Yue Qi, Yu-wen Hung, Thomas Girke, David Ann, Victoria Seewaldt, Ernest Martinez. MYC acetylated lysine residues drive oncogenic cell transformation and regulate select genetic programs for cell adhesion-independent growth and survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7072.

Abstract 6587: Discovery of molecular glue of FBXW7:MYC interaction using small molecule microarray

Abstract MYC deregulation is a key driver and feature of many human cancers. Yet, despite its importance in cancer progression, MYC is considered “undruggable” by small molecules due to its disordered conformation. Therefore, interfering with its interactions with protein partners to modulate MYC activity appears viable. FBXW7 is an E3 ligase, which serves as a constituent of the SCF (Skp1-Cul1-F box) ubiquitin ligase complex. It mediates the degradation of both c-Myc and Cyclin E through ubiquitin-dependent proteolysis. FBXW7 mutations are found in various human tumor types, establishing its role as a general human tumor suppressor. Specifically, FBXW7 down-regulation increases MYC level, and its expression correlates to lethal outcomes of breast epithelial cells, poor prognosis in glioblastoma, and T cell acute lymphoblastic leukemia remission. Therefore, our project aims to discover potent small molecule glues that would enhance the interaction between FBXW7 and MYC, leading to MYC degradation through proteolysis and eventually suppression of various cancers. Taking advantage of our high throughput screening technology, Small Molecule Microarrays (SMMs), we successfully identified 29 compounds that potently bind to both c-Myc and FBXW7 after screening our 65,000 ‘drug-like’ small molecule library. We then performed secondary assays using a mCherry-GFP MYC reporter cell line to identify our lead compound, KI-FBX-001, which decreases MYC’s expression:transcription ratio by over 50% compared to control. Additionally, this down-regulation can be rescued by protease inhibitor MG132, indicating that the effect is achieved via proteolysis. In vitro dose-response and time-course treatment revealed an optimal drugging concentration at 15uM in K562 cells, with inhibition reaching its peak at the 4-hour time point at over 60%. Using the Qiagen MYC reporter assay, the in vitro IC50 of KI-FBX-001 is found at 7.42uM. Beyond the protein level, the qPCR result also reveals a decrease in MYC mRNA level at over 70%. Surface Plasmon Resonance (SPR) is used to confirm direct binding between FBXW7 and KI-FBX-001, which showed promising binding with kd=0.17µM. Proximity Ligation Assay further confirms KI-FBX-001’s effect in enhancing FBXW7:MYC interaction in the cell. In summary, our findings proved that KI-FBX-001 is a promising molecular glue in the FBXW7:MYC interaction, and can effectively modulate the MYC level for future therapeutic purposes. Citation Format: Shenghao Guo, Morgan Stilgenbauer, Bocheng Wu, Yatin Mankan, Angela N. Koehler. Discovery of molecular glue of FBXW7:MYC interaction using small molecule microarray [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6587.

Abstract 2968: TWIST1-induced suppression of oncogene-induced senescence in non-small cell lung cancer requires the transactivation domain of TWIST1

Abstract Non-small cell lung carcinoma (NSCLC) is a major cause of cancer mortality. High expression of the epithelial-to-mesenchymal transition transcription factor TWIST1 is strongly associated with metastatic cancers and treatment resistance. Additionally, TWIST1 can upregulate O-GlcNAcylation which (1) is required to suppress fail-safe programs such as oncogene (KRasG12D)-induced senescence (OIS) to accelerate tumorigenesis in primary NSCLC tumors, and (2) is a potential modulator of DNA repair/radiation response. To decipher the domains and transcriptional targets required for tumorigenicity and radioresistance, we created a novel genetically engineered mouse model (GEMM) allowing tetracycline-inducible expression in the lung epithelium (via lung specific CCSP-reverse tetracycline transactivator (C)) of KRasG12D (R) with Twist1wt (T) or with Twist1F191G transactivation-null mutant (F). CRT mice had shorter tumor-free survival and more aggressive tumors compared to CR/CRF mice indicating that the Twist1 transactivation domain is required for Twist1-dependent tumorigenesis acceleration. Also, Twist1wt expression promoted radioresistance in cell lines and GEMMs. Contrary to CRT, CRF showed a progressive loss of Twist1F191G expression over time suggesting no functionality/no selective advantage. CRT lung tumors had higher proliferation (Ki67) and lower cell-cycle arrest (p16) compared to CR/CRF suggesting that the transactivation domain of Twist1 is important for the suppression of OIS. Supporting these data, we observed in non-cancer Human Bronchial Epithelial Cell (HBEC) that the co-expression of human TWIST1wt (HBEC-TWIST1wt) could suppress HRasG12V-induced senescence while the transactivation-null TWIST1F187G mutant (HBEC-TWIST1F187G) could not. Additionally, HBEC expressing HRasG12V-TWIST1wt had enhanced tumorigenic/invasive programs. Interestingly, we observed that the inhibition of O-GlcNAcylation rescued OIS in HBEC-HRasG12V-TWIST1wt while the stimulation of O-GlcNAcylation in HBEC-HRasG12V-TWIST1F187G suppressed OIS. Furthermore, TWIST1wt expression with HRasG12V modulated MYC downstream targets and the inhibition of MYC activity using the novel MYC inhibitor MYCi975 in HBEC-HRasG12V-TWIST1wt also rescued OIS induction. Altogether, these results suggest that TWIST1 may suppress OIS via MYC signaling and nominate MYCi975 as a means to activate latent OIS programs. In this context, MYC inhibiting strategies could serve as a therapeutic sensitizer for TWIST1-positive NSCLC. This work and our future studies on TWIST1 toward the control of OIS, O-GlcNAcylation and of mechanisms of radioresistance may help to identify new potential NSCLC therapeutic strategies. Citation Format: Audrey M. Lafargue, Hailun Wang, Sivarajan T. Chettiar, Rajendra P. Gajula, Amol C. Shetty, Yang Song, Brian W. Simons, Triet Nguyen, Christine Lam, Francesca A. Carrieri, Caleb Smack, Nick Connis, Dipanwita Dutta Chowdhury, Jinhee Chang, Danielle Council, Katriana Nugent, Ismaeel Siddiqui, Kekoa Taparra, Mohammad Rezaee, Natasha Zachara, Zachary S. Morris, Christopher McFarland, Sarki Abba Abdulkadir, Christine L. Hann, Phuoc T. Tran. TWIST1-induced suppression of oncogene-induced senescence in non-small cell lung cancer requires the transactivation domain of TWIST1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2968.

Abstract 7166: Pharmacokinetic analysis of omomyc therapeutic protein in preclinical and human tumor tissue using targeted mass spectrometry

Abstract MYC is a pleiotropic transcription factor consisting in three paralogs which coordinate gene expression and regulates cell proliferation, cell differentiation, cell cycle, metabolism and apoptosis. The 3 paralogs (c-, N-, and L-MYC) have similar biological functions but show distinct tissular and timing expression patterns. To function, MYC must heterodimerize with its partner MAX and bind DNA via its basic, helix-loop-helix, leucine zipper domain (B-HLH-LZ). It has been estimated that MYC is aberrantly expressed in up to 70% of human cancers and hence MYC has been termed a “most wanted target for cancer treatment”. However, due to its nuclear localization, its intrinsic disorder and lack of an enzymatically active site, development of clinical grade potent & selective small molecules targeting MYC remains an obstacle. Omomyc is a 91-amino-acids-long polypeptide that derives from the B-HLH-LZ domain of human MYC, containing four amino acid substitutions. Omomyc is capable of forming homodimers and heterodimers with MYC and with MAX and such dimers blunt MYC activity by preventing its access to target DNA sequences (E-boxes) and replacing it by inactive complexes. Previous studies showed the cell-penetrating properties of recombinant Omomyc and its subsequent nuclear localization, where it displaces MYC from its genomic locations, causing a specific shutdown of MYC transcriptional signature and effectively stopping cell cycle progression. Further in vivo preclinical experiments showed that Omomyc blocks MYC-driven transcription and proliferation, induces apoptosis and recruitment of intratumoral immune infiltrates, overall significantly abrogating tumor progression and reducing tumor grading. A targeted LC-MS/MS assay for the absolute quantification of 4 Omomyc proteotypic peptides was developed to assess the structural integrity of Omomyc and quantify functional Omomyc in tumor tissue. LLOQ and linear range of the panel was determined by spiking in dilution series of recombinant Omomyc protein into preclinical tumor tissue lysates. Finally, in the context of Omomyc clinical trial (NCT04808362), we used the panel to for quantification of functional Omomyc in FFPE tumor biopsies from 18 patients who received i.v. injection of Omomyc therapeutic. Data show that 2 h after i.v. administration, higher concentrations of the drug are reached in the tumors compared with serum and persist there with at least one order of magnitude higher concentration than in serum 72 h after administration in preclinical models. Functional Omomyc protein could be quantified in all of the 18 clinical tumor biopsies from treated patients showing that characterized LC-MS/MS proteomic assay can be used in the normal routine of a clinical trial for accurate quantification of therapeutic miniproteins. Citation Format: Marie-Eve Beaulieu, Sandra Martinez-Martin, Maik Mueller, Yuehan Feng, Jakob Vowinckel, Antoine Amaury Lachaud, Laura Soucek. Pharmacokinetic analysis of omomyc therapeutic protein in preclinical and human tumor tissue using targeted mass spectrometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7166.

Manipulating Myc for reparative regeneration.

The Myc family of proto-oncogenes is a key node for the signal transduction of external pro-proliferative signals to the cellular processes required for development, tissue homoeostasis maintenance, and regeneration across evolution. The tight regulation of Myc synthesis and activity is essential for restricting its oncogenic potential. In this review, we highlight the central role that Myc plays in regeneration across the animal kingdom (from Cnidaria to echinoderms to Chordata) and how Myc could be employed to unlock the regenerative potential of non-regenerative tissues in humans for therapeutic purposes. Mastering the fine balance of harnessing the ability of Myc to promote transcription without triggering oncogenesis may open the door to many exciting opportunities for therapeutic development across a wide array of diseases.

MYC activity at enhancers drives prognostic transcriptional programs through an epigenetic switch.

The transcription factor MYC is overexpressed in most cancers, where it drives multiple hallmarks of cancer progression. MYC is known to promote oncogenic transcription by binding to active promoters. In addition, MYC has also been shown to invade distal enhancers when expressed at oncogenic levels, but this enhancer binding has been proposed to have low gene-regulatory potential. Here, we demonstrate that MYC directly regulates enhancer activity to promote cancer type-specific gene programs predictive of poor patient prognosis. MYC induces transcription of enhancer RNA through recruitment of RNA polymerase II (RNAPII), rather than regulating RNAPII pause-release, as is the case at promoters. This process is mediated by MYC-induced H3K9 demethylation and acetylation by GCN5, leading to enhancer-specific BRD4 recruitment through its bromodomains, which facilitates RNAPII recruitment. We propose that MYC drives prognostic cancer type-specific gene programs through induction of an enhancer-specific epigenetic switch, which can be targeted by BET and GCN5 inhibitors.

Clinical and genomic characterization of chemoradiation-resistant HPV-positive oropharyngeal squamous cell carcinoma.

Most patients with HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) have an excellent response to chemoradiation, and trials are now investigating de-escalated treatment. However, up to 25% of patients with HPV-positive OPSCC will experience recurrence, and up to 5% will even progress through primary treatment. Currently, there are no molecular markers to identify patients with poor prognosis who would be harmed by de-escalation. Herein we report the clinical and genomic characteristics of persistent HPV-positive OPSCC after definitive platinum-based chemoradiation therapy. Patients with HPV-positive OPSCC treated with curative intent platinum-based chemoradiation between 2007 and 2017 at two institutions and with a persistent locoregional disease were included. We evaluated clinical characteristics, including smoking status, age, stage, treatment, and overall survival. A subset of five patients had tissue available for targeted exome DNA sequencing and RNA sequencing. Genomic analysis was compared to a previously published cohort of 47 treatment-responsive HPV+ OPSCC tumors after batch correction. Mutational landscape, pathway activation, and OncoGPS tumor states were employed to characterize these tumors. Ten patients met the inclusion criteria. The tumor and nodal stages ranged from T1 to T4 and N1 to N2 by AJCC 8th edition staging. All patients were p16-positive by immunohistochemistry, and eight with available in situ hybridization were confirmed to be HPV-positive. The 1-year overall survival from the time of diagnosis was 57%, and the 2-year overall survival was 17%. TP53 mutations were present in three of five (60%) persistent tumors compared to 2% (one of 47) of treatment-responsive HPV-positive tumors (p = 0.008). Other genes with recurrent mutations in persistent HPV-positive OPSCC tumors were NF1, KMT2D, PIK3C2B, and TFGBR2. Compared to treatment-responsive HPV-positive tumors, persistent tumors demonstrated activation of DNA Repair and p53, EMT, MYC, SRC, and TGF-beta signaling pathways, with post-treatment samples demonstrating significant activation of the PI3K-EMT-Stem pathways compared to pretreatment samples. Chemoradiation-resistant HPV-positive OPSCC occurs infrequently but portends a poor prognosis. These tumors demonstrate higher rates of p53 mutation and activation of MYC, SRC, and TGF-beta pathways. A comparison of tumors before and after treatment demonstrates PI3K-EMT-Stem pathways post-treatment in HPV-positive tumors with persistent disease after platinum-based chemoradiation.

Abstract P27: Proteogenomic and metabolomic analysis of acute myeloid leukemia reveals molecular and functional underpinnings of cellular and clinical phenotypes

Abstract Acute myeloid leukemia (AML) is a blood malignancy of poor prognosis with marked heterogeneity. To elucidate the underlying mechanisms that drive AML as part of the Clinical Proteomic Tumor Analysis Consortium (CPTAC) effort, we performed large scale comprehensive genomics, transcriptomics, proteomics including multiple post-translational modifications (phosphorylation, acetylation, and glycosylation), metabolomics, and lipidomics characterization of 173 treatment-naïve AML patients. Applying the similarity network fusion method on both transcriptomics and proteomics data, we identified 8 proteogenomic clusters. These clusters recapitulate specific recurrent mutations, fusions, structural variants, and established clinical subtypes available within the cohort, as well as reveal new cluster-specific phenotypes within other multi-omic datasets. We used single-cell RNAseq data as a reference to perform immune component analysis for collected bulk samples. The result reveals that our proteogenomic clustering also captures the variations of AML differentiation hierarchies including CD14+ monocyte-like and GMP-like AML. To assess the complex disease nature of AML, we performed functional analysis for each cluster to reveal interplay between multiple genomic aberrations such as NPM1, FLT3-ITD, DNMT3A mutations, complex chromosomal alterations, and the leukemia cell differentiation. Additionally, the multi-omics analysis performed not only connects previously identified molecular drivers and cell differentiation variations within AML, but also links them with observed cancer metabolomic reprogramming alongside differences in MTOR signaling, MYC activities, mitochondrial activities, and drug responses. Moreover, our study also identified site-specific post-translational modifications previously not known in AML, highlighting the valuable insights and clinical relevance of these newly identified clusters. Citation Format: Shih-Chun Alec Chu, Yi Hsiao, Yamei Deng, Chenwei Wang, Jennifer Kyle, Yongchao Dou, James Pino, Camilo Posso, Leanne Henry, Ginny Li, Li Ding, Lijun Chen, Mamie Lih, Yifat Geffen, Gilbert Omenn, Chandan Kumar, Saravana Dhanasekaran, Fengchao Yu, Elie Traer, Jeffrey W. Tyner, Hui Zhang, Tao Liu, Sara Gosline, Bing Zhang, Arul Chinnaiyan, Alexey I Nesvizhskii, Marcin Cieslik. Proteogenomic and metabolomic analysis of acute myeloid leukemia reveals molecular and functional underpinnings of cellular and clinical phenotypes [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P27.