High Levels Of MYC Research Articles

Abstract B019: Clinical and molecular correlates of circulating tumor DNA (ctDNA) dynamics in breast tumors resistant to neoadjuvant therapy (NAT)

Abstract Purpose: We examined the utility of serial ctDNA testing for refining risk stratification of NAT- resistant tumors (RTs, defined as residual cancer burden, RCB-II/III) and predicting early treatment response. We also characterized the molecular correlates of ctDNA dynamics in RTs to elucidate the mechanisms associated with increased metastatic potential. Methods: We performed serial ctDNA testing using a tumor-informed personalized assay (SignateraTM, Natera Inc.) in 723 patients (pts) with high-risk early-stage breast cancer receiving NAT (I-SPY2). Tumors were stratified according to RCB class and ctDNA dynamics (clearance patterns), and the distant recurrence-free survival (DRFS) between groups was compared. We examined the predictive value of ctDNA dynamics using the RCB index (the continuous measure of RCB) as the response endpoint. Serial whole exome sequencing (WES), gene expression profiling (GEP), and reverse-phase protein analysis (RPPA) data were analyzed to elucidate the molecular biology of RTs. Results: Of the 723 pts, 300 (41%) had hormone receptor (HR)+HER2-, 237 (33%) triple-negative (TN), and 186 (26%) HER2+ breast cancers with pretreatment ctDNA-positivity rates of 76%, 92%, and 77%, respectively. 321 (45%) pts had responding tumors (RCB-0/I), and 55% had RTs: RCB-II (n=255) and RCB-III (n=132). CtDNA dynamics revealed heterogeneity in the metastatic potential of RTs, including tumors with a reduced propensity to metastasize (persistent ctDNAneg and early clearance). Failure to clear ctDNA after NAT was associated with an increased risk of metastatic recurrence and death (3-yr DRFS rates: no clearance=35% vs. persistent ctDNAneg=98%, p<0.001). Early clearance of ctDNA, 3 weeks after treatment initiation, skewed the distributions toward lower RCB indices (favorable response), including in arms containing immune checkpoint inhibitors. Of the 387 RTs, 316 had ctDNA data post-NAT, of which only 51 (16%) were ctDNA+. We hypothesized that changes in the mutational landscape during NAT could have impacted the detection of patient-specific ctDNA targets selected from WES of pretreatment tumors. However, serial WES (n=100) of RTs showed that these targets remained detectable in solid tissue over time, even in persistent ctDNAneg. To investigate the biology of persistent ctDNA positivity, we used logistic regression to analyze serial GEP (n=174) and RPPA (n=98) data in RTs that were ctDNA+ at pretreatment, comparing those with ctDNA clearance by surgery vs. those that remained ctDNA+. We found subtype- specific associations: In HR+HER2-, failure to clear ctDNA during NAT was associated with low pretreatment ER/AR, high early on-treatment proliferation, and low HER2-signaling after NAT, while in TN, fewer associations were observed; these included high pretreatment levels of MYC and proliferation. Conclusions: ctDNA dynamics refined risk stratification of RTs and enabled early prediction of treatment response. Understanding the molecular profiles of RTs based on ctDNA dynamics could aid treatment selection to overcome resistance to NAT. Citation Format: Mark Jesus M. Magbanua, Denise M. Wolf, Samuel Rivera-Hinojosa, Ziad Ahmed, Nayelis A. Manon, Rosalyn Sayaman, Antony Tin, Ekaterina Kalashnikova, Lamorna Brown Swigart, Gillian L. Hirst, Christina Yau, Wen Li, Claudine Isaacs, Rebecca A. Shatsky, Amy L. Delson, Charuta C. Palsuledesai, Angel Rodriguez, Minetta C. Liu, Paula R. Pohlmann, Laura J. Esserman, Hope S. Rugo, Angela DeMichele, Laura van 't Veer. Clinical and molecular correlates of circulating tumor DNA (ctDNA) dynamics in breast tumors resistant to neoadjuvant therapy (NAT) [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B019.

Chromosome 8q24 amplification associated with human hepatocellular carcinoma predicts MYC/ZEB1/MIZ1 transcriptional regulation.

Genomic instability is associated with late stage carcinomas and the epithelial mesenchymal transition (EMT). Of note is chromosome 8q24 amplification that has been documented in many epithelial-derived carcinomas. On this amplified region is the potent oncogene, c-myc. Not only does MYC overexpression activate targets that promote cell proliferation, it also activates transcription factors that drive EMT, including ZEB1. Further reinforcing EMT, overexpressed MYC also represses tumor suppressors involved in promoting the epithelial phenotype, including MIZ1. We predict that as carcinomas progress, chromosome 8q24 is amplified leading to high MYC levels that leads to ZEB1 expression and MIZ1 repression driving cells through EMT. To interrogate this clinically, limited cohorts of human epithelial-derived carcinomas were examined for MYC/ZEB1/MIZ1 expression patterns across increasing carcinoma grades. Interestingly, the predicted temporal patterns were only observed in hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinomas. Yet MIZ1 proved to be an excellent marker to assess carcinoma progression across types. We expanded the HCC cohort and determined that c-myc amplification was restricted to grade III/IV HCC that also exhibited increased MYC and ZEB1 nuclear expression whereas cytosolic MIZ1 expression was lost and only nuclear expression retained. These same resections were obtained from only individuals who had histories of alcohol consumption that were also diagnosed with cirrhosis, metastasis and had viral hepatitis suggesting etiology-specific mechanisms of cancer progression. Finally, analysis performed in Hep3B cells determined that alterations in MYC expression promoted the predicted changes in ZEB1 and MIZ1 expression and/or distributions and in markers for EMT further suggesting a relationship among these three transcription factors in HCC and their correlation to driving EMT.

MDB-109. CHARACTERIZING DISTINCT SIGNATURES OF RNA TRANSLATIONAL REGULATION UPON MYC OVEREXPRESSION IN H9 NEURAL STEM CELLS

Abstract BACKGROUND Medulloblastoma (MB), a childhood brain tumor, emerges typically between 6-8 years, originating from cerebellar neuronal stem cells. Recurrent genetic alterations, notably MYC amplifications, are associated with poor therapy response. Subgrouped as WNT-MB, SHH-MB, Group 3 MB, and Group 4 MB, Group 3 MB is characterized by high-level MYC amplification in approximately 17% of cases. Patients with Group 3 MB and MYC activation present distinct transcriptional and proteomic signatures, including elevated levels of ribosomal proteins and ribosome assembly proteins. METHODS We conducted a comprehensive analysis of RNA-seq and Ribo-seq in human neural stem cells (H9 NSCs) with amplified MYC expression. H9-NSCs were cultured in tumor stem media and transduced with lentivirus for either GFP control or MYC cDNA. RNA and ribosome-protected fragments were isolated from cell lysates for RNA-seq and Ribo-seq in biological triplicates. Ribo-seq data was analyzed using ORFquant for coding sequence quantification and deltaTE for translational efficiency. RESULTS Overexpressing MYC in H9-NSCs led to significant differential expression of 4194 genes (p adj < 0.05) using RNA-seq and 2860 genes (p adj < 0.05) using Ribo-seq. While RNA-seq and Ribo-seq exhibited high correlation for most genes, we identified 364 differentially translated genes (p adj < 0.05) upon MYC overexpression. Genes regulated solely at the translational level were enriched for functions in cytoplasmic translation initiation, viral translation, and response to interleukin-4. Key genes highly regulated at the Ribo-seq level included CCND3, YBX1, and EEF2. Validating with proteomics data from the CPTAC medulloblastoma tissue set confirmed protein-specific upregulation of these targets in MYC-driven Group 3 medulloblastomas. CONCLUSIONS MYC overexpression revealed distinct behaviors in differentially expressed genes, either regulated at the level of translation alone, correlated with transcription, or independent of transcription. We anticipate that our findings will contribute to a refined understanding of the molecular landscape of MB, particularly in the context of Group 3 MB.

Mitotic gene regulation by the N-MYC-WDR5-PDPK1 nexus

BackgroundDuring mitosis the cell depends on proper attachment and segregation of replicated chromosomes to generate two identical progeny. In cancers defined by overexpression or dysregulation of the MYC oncogene this process becomes impaired, leading to genomic instability and tumor evolution. Recently it was discovered that the chromatin regulator WDR5—a critical MYC cofactor—regulates expression of genes needed in mitosis through a direct interaction with the master kinase PDPK1. However, whether PDPK1 and WDR5 contribute to similar mitotic gene regulation in MYC-overexpressing cancers remains unclear. Therefore, to characterize the influence of WDR5 and PDPK1 on mitotic gene expression in cells with high MYC levels, we performed a comparative transcriptomic analysis in neuroblastoma cell lines defined by MYCN-amplification, which results in high cellular levels of the N-MYC protein.ResultsUsing RNA-seq analysis, we identify the genes regulated by N-MYC and PDPK1 in multiple engineered CHP-134 neuroblastoma cell lines and compare them to previously published gene expression data collected in CHP-134 cells following inhibition of WDR5. We find that as expected N-MYC regulates a multitude of genes, including those related to mitosis, but that PDPK1 regulates specific sets of genes involved in development, signaling, and mitosis. Analysis of N-MYC- and PDPK1-regulated genes reveals a small group of commonly controlled genes associated with spindle pole formation and chromosome segregation, which overlap with genes that are also regulated by WDR5. We also find that N-MYC physically interacts with PDPK1 through the WDR5-PDPK1 interaction suggesting regulation of mitotic gene expression may be achieved through a N-MYC-WDR5-PDPK1 nexus.ConclusionsOverall, we identify a small group of genes highly enriched within functional gene categories related to mitotic processes that are commonly regulated by N-MYC, WDR5, and PDPK1 and suggest that a tripartite interaction between the three regulators may be responsible for setting the level of mitotic gene regulation in N-MYC amplified cell lines. This study provides a foundation for future studies to determine the exact mechanism by which N-MYC, WDR5, and PDPK1 converge on cell cycle related processes.

Abstract 561: Targeting Y box binding protein 1 (YBX1) in MYC-amplified medulloblastoma

Abstract Medulloblastoma is the most common malignant pediatric brain tumor. Patients diagnosed with MYC-amplified group 3 medulloblastoma (G3 MB) have a devastating prognosis with a 5-year survival rate of <50%. Standard of care (SOC) consists of maximal resection surgery followed by chemoradiotherapy, but these extensive treatments leave patients with neurocognitive and developmental deficits. Therefore, the identification of novel targets that can be used alone or in combination with SOC is of utmost importance. Using a batch-normalized bulk RNA-seq dataset (GSE124814) comprised of 1350 MB samples and 291 normal cerebella, we reveal upregulation of Y-box binding protein 1 (YBX1) in MB compared to normal cerebellum. Further analysis of this dataset and others demonstrates G3 tumors that display high MYC levels also have the highest YBX1 expression. Analysis of ChIP-seq data shows strong binding of MYC to the YBX1 promoter in G3 MB, concurrent with H3K27ac and H3K4me3 histone modifications. Using a well-established ex vivo model, we demonstrate increased YBX1 expression as normal cerebellar progenitor cells transition to tumorigenic G3 MB stem-like cells. Recent work in leukemia reveals a reciprocal regulation of MYC by YBX1, however, this mechanism is not well established in G3 MB. We utilized an orthogonal pharmacogenetic approach to inhibit YBX1 function and determine its effects on G3 MB tumor biology. Suppression of YBX1 significantly reduced the proliferation and decreased MYC protein expression of G3 MB cells in vitro. These findings support a reciprocal MYC-YBX1 axis, which could influence tumor growth in vivo. Orthotopic implantation of YBX1 KO G3 MB cells leads to increased animal survival. We next evaluated the therapeutic potential of two recently characterized YBX1 inhibitors which display single agent efficacy in vitro and in vivo. YBX1 inhibitors synergized with SOC in vitro lowering the IC50 for both chemo and radiotherapy, which could reduce adverse effects experienced by patients. In silico molecular docking using PyMOL predicts YBX1 inhibitors interact with amino acids within the cold shock domain of YBX1, likely impairing its ability to bind RNA. We performed formaldehyde RNA immunoprecipitation (fRIP)-sequencing to identify changes in RNA transcript levels following YBX1 pharmacological inhibition. Disruption of YBX1-RNA binding impacts the stability of known oncogenic transcripts which may be required for tumorigenesis. Our findings impart an alluring prospect of targeting YBX1 in combination with standard-of-care, and possibly other compounds to improve patients’ outcomes. Citation Format: Stephen Carney, John Hemenway, Ashmitha Rajendran, Matt Hathaway, Eric Nealy, Christian Bonatto, Dhanir Tailor, Sanjay Malhotra, Myron Evans. Targeting Y box binding protein 1 (YBX1) in MYC-amplified medulloblastoma [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 561.

Accurate Measurement of Cell Number-Normalized Differential Gene Expression in Cells Treated With Retinoic Acid.

Genome-wide gene expression analysis is a commonly used method to quantitatively examine the transcriptional signature of any tissue or cell state. Standard bulk cell RNA sequencing (RNA-seq) quantifies RNAs in the cells of the tissue type of interest through massive parallel sequencing of cDNA synthesized from the cellular RNA. The subsequent analysis of global RNA expression and normalization of RNA expression levels between two or more samples generally assumes that cells from all samples produce equivalent amounts of RNA per cell. This assumption may be invalid in cells where MYC or MYCN expression levels are markedly different and thus, overall mRNA expression per cell is altered. Here, we describe an approach for RNA-seq analysis of MYCN-amplified neuroblastoma cells during treatment with retinoic acid, which causes dramatic downregulation of MYCN expression and induces growth arrest and differentiation of the cells. Our procedure employs spiked-in RNA standards added in ratio to the number of cells in each sample prior to RNA extraction. In the analysis of differential gene expression, the expression level of each gene is standardized to the spiked-in RNA standard to accurately assess gene expression levels per cell in conditions of high and low MYCN expression. Our protocol thus provides a step-by-step experimental approach for normalizing RNA-seq expression data on a per-cell-number basis, allowing accurate assessment of differential gene expression in cells expressing markedly different levels of MYC or MYCN. Key features • High levels of MYC and MYCN expression in cancer cells cause substantial increases in the levels of overall mRNA expression per cell. • RNA-seq using control RNAs spiked-in on a per-cell basis more accurately reflects global expression changes, when comparing cell populations with substantially different MYCN expression levels. • In MYCN-amplified neuroblastoma, retinoic acid dramatically decreases MYCN expression levels, resulting in large changes in overall RNA expression levels per cell.

Changes in spindle morphology driven by TPX2 overexpression in MYC-driven breast cancer cells.

The MYC oncogene was previously shown to induce mitotic spindle defects, chromosome instability, and reliance on the microtubule-associated protein TPX2 to survive, but how TPX2 levels affect spindle morphology in cancer cells has not previously been examined in detail. We show that breast cancer cell lines expressing high levels of MYC and TPX2 possess shorter spindles with increased TPX2 localization at spindle poles. A similar effect was observed in non-transformed human RPE-1 cells compared to a tumor cell line (HeLa) that overexpresses MYC . These results demonstrate that TPX2 alters spindle length and morphology in cancer cells, which may contribute their ability to divide despite MYC-induced mitotic stress.

MYC amplifications are a common event in urothelial bladder carcinomas associated with an aggressive tumor phenotype

Abstract Introduction/Objective Gene amplifications of the proto-oncogen MYC are common events in carcinomas. In this study, we analyzed the impact of MYC amplifications on tumor aggressiveness and patient prognosis in urothelial bladder carcinomas. Methods/Case Report MYC copy number status was analyzed on more than 2,500 urothelial bladder carcinomas in a tissue microarray format by using dual-labeling fluorescence in-situ hybridization (FISH) with probes for centromere 8 and MYC (8q24). The results were compared with tumor phenotype and patient outcome. Results (if a Case Study enter NA) MYC amplification (ratio MYC/centromere 8 ≥2) occurred in 10% of 2,052 analyzable urothelial bladder carcinomas. Of these, 141 (6.8%) had a low level MYC copy number gain (MYC/centromere 8 ratio ≥2 and ≤ 3) and 68 (3.3%) had high-level MYC amplifications (MYC/centromere 8 ratio >3) . The rate of MYC gain and MYC amplification increased from pTa G2 low (0.6%), to pTa G2 high (7.7%), and pTa G3 (8.2%) carcinomas, and were highest in muscle-invasive pT2-4 (13.2%) carcinomas (p<0.0001). In muscle-invasive urothelial carcinomas, the MYC amplification rate increased from pT2 (13.1%), to pT3 (14.7%), and pT4 (19.8%) but these differences failed to reach statistical significance (p=0.0902). MYC amplification was also unrelated to patient survival in 526 patients with pT2-4 urothelial carcinomas who had undergone cystectomy (p=0.5526). Conclusion MYC amplification is a common event in urothelial bladder carcinoma - especially in case of pTa G3 and muscle-invasive carcinoma. The association of MYC amplification with advanced stage may reflect increasing genomic instability going along with tumor progression.

Cooperative Networks between MYC and XPO1 Associated with Decreased T-Cell Presence and a Depleted Tumor Microenvironment May be Addressed By the Synergistic Combination of AZD4573 and Selinexor

Background: Genomically-targeted Diffuse Large B-cell (DLBCL) treatments for refractory or relapsing patients remain a clinical need alongside the growing success of Chimeric Antigen T-cells (CAR-T), which have ushered in durable responses in 40-70% of patients. However, recent studies have identified factors indicative of an inferior CAR-T response. Recent tumor Ecotype studies from Kotlov and Steen reveal that a Depleted or Cold tumor microenvironment (TME) is associated with poor CAR-T response. Notable features of these environments include a decreased presence of native T-cells alongside oncogenic alterations associated with their exhaustion, namely MYC and XPO1. Few precision treatments exist given the difficulty targeting MYC, but two avenues have shown recent success: inhibition of the downstream oncogene CDK9 via AZD4573 and upstream inhibition of a MYC propagator, the nuclear exportin XPO1 via Selinexor. Herein, we report the first observations of synergy between AZD4573 and Selinexor within DLBCL cell lines and data supporting that these genes may serve as accessible markers for inferior tumor immune microenvironments. Methods: We combined progression vs. response results from four previous CAR-T genomics studies, noting MYC status (Jaeger 2020, Jain 2021, Sworder 2021, and Shouval 2022). We next analyzed a 418-patient de-novo DLBCL patient cohort (Xu-Monette 2020) and 48-patient TCGA cohort after GEDIT immune deconvolution for differential expression and component enrichment between patients expressing high levels of MYC and XPO1 (+1 Standard Deviation) compared to normal counterparts. Significantly expressed genes were evaluated via gene set enrichment analyses. We next assayed AZD4573 and Selinexor vs. 7 DLBCL cell lines, 5 of which harbor MYC alterations, across a series of 9 concentrations (78nM-20µM) across 3-9 replicates. We followed these results by combining treatments in the MYC-altered Ly3, DHL6, and DHL4 cell lines, analyzing results using the BLISS synergy model. Results: Integrative analyses support that MYC alterations are associated with significantly reduced CAR-T outcomes. Of the 93 MYC-altered patients within the total 246-patient cohort, 65.59% of them experienced progression during CAR-T treatment compared to just 50.33% of WT CAR-T patients (p = 0.0242). Differential gene expression analyses between de-novo patients expressing high MYC or XPO1 revealed losses in Cytokine Signaling pathways (FDR = 6.96E-18) and gains in Cell Cycle modulators (FDR = 2.525E-20). Importantly, CD8A expression was significantly lower in these patients (FDR < 0.0001) alongside other key reductions of CDKN1A and TNFAIP3. After immune deconvolution, both High-MYC and High-XPO1 patients displayed significant losses of CD8 T-cell association, respectively (FDR = 0.0005, FDR = 0.0396). Notably, High-MYC patients without a CD8 T-cell presence experienced an inferior survival prognosis compared to High-MYC counterparts with a CD8 presence (p = 0.0272). High MYC or XPO1 patients that experienced a failure event before 24-months (EFS24) had significantly lower activated CD4 T-cell presence compared to normal counterparts as well (FDR = 0.0095, FDR = 0.0069). Next, AZD4573 and Selinexor assays vs. DLBCL cell lines demonstrated dose and MYC-dependent cell inhibition, with ED50 values ranging between 58.1 nM to 8.07 µM. Lastly, the molecules were combined vs. Ly3, DHL6 and DHL4 cell lines, resulting in synergistic reduction of cell viability and BLISS synergy scores of 33.76, and 11.86, and 5.04, respectively. Conclusions: Our results support that the cooperation between MYC and XPO1 is associated with T-cell reductions characteristic of a Depleted or Cold TME, a key issue for CAR-T success. We applied the targeted therapies AZD4573 and Selinexor vs. cell line models to address this pathway, with both displaying anti-tumor effects as single agents. Most importantly, we observed synergistic activity in the MYC-harboring Ly3 and DHL6 cell lines, and in the WT DHL4 cell line when these molecules were combined. These results are supported by integrative analyses that highlight the importance of targeting aberrant MYC signaling via CDK9 and XPO1 inhibition, revealing a potential avenue to address inferior response rates faced by patients harboring MYC-positive, Depleted tumor microenvironments that would otherwise face an inferior CAR-T prognosis.

Pax5 Heterozygosity Affects B-Cell Differentiation and Generates a Deregulated Precursor Population in the Bone Marrow

Introduction: PAX5 is a well-known master regulator for B-cell development, commitment and identity. Hence, germline or somatic deregulation of PAX5 facilitates the development of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Previously, our group has described B-cell development abnormalities in three families carrying PAX5 germline variants ( Escudero et al., Leukemia, 2022) as well as established a link between Pax5 heterozygosity and infection exposure, thereby recapitulating the incomplete penetrance of the human BCP-ALL ( Martin-Lorenzo et al., Cancer Discovery, 2015). Nevertheless, the molecular single-cell characteristics of the susceptible precursor population mediated by reduced Pax5 levels is still poorly understood. Methods: Here, we deeply characterized the susceptible precursor population in Pax5+/- mice utilizing single-cell RNA Sequencing (scRNA-Seq), multicolor flow cytometry analyses and in-vivo transplantation models. Results: Our data show, that Pax5 heterozygosity leads to an aberrant pre-BII population (B220 +CD19 +IgM -CD25 +) in the bone marrow (BM) of Pax5+/- mice (p=0.0026, Student's t-test) (Fig. 1A). This population is stable over time and characterized by higher CD25 and IL-7-Receptor expression (p<0.0001, Student's t-test). Furthermore, bulk RNA-Sequencing of the pre-BII population revealed B-cell receptor (BCR) signaling as one of the top downregulated pathways (including CD79a/b, Lyn and CD72), while DNA replication and cell cycle signaling pathways were upregulated in Pax5+/- vs. Wildtype (WT) pre-BII cells. ScRNA-Seq analyses confirmed these results (Fig. 1B) and further showed that pre-BII cells of Pax5+/- mice are skewed toward the less commonly used lambda light chain BCR-rearrangements. The observed delay in the transition to IgM-positivity was additionally validated in a murine in-vivo transplantation model. Here, after 72 hours pre-BII cells from Pax5+/- mice preferably homed to the BM, while matured transplanted WT cells were predominantly found in the spleen (p=0.0023 and p=0.0140, respectively, Student's t-test). To depict the full spectrum of malignant transformation based on the described deregulated precursor B-cell population, we additionally performed scRNA-Seq on different stages of Pax5-mediated BCP-ALL evolution. Our data show that in a first step, arrested pre-leukemic cells of Pax5+/- mice lose their B-cell identity and display high Myc levels. Sequentially, full blown BCP-ALL arises in a second step after acquisition of additional oncogenic driver mutations (including Jak1 and Jak3). Interestingly, although the analyzed BCP-ALLs of Pax5+/- mice were all based on the same predisposition, they greatly varied in their transcriptional profile and cell-cycle state, depending on their oncogenic 2 nd hit. Conclusion: In summary, we deeply characterized how reduced Pax5 transcriptional activity in the BM generates a predisposed precursor B-cell environment, which is susceptibility for malignant transformation. These findings are important for understanding the molecular mechanisms to prevent or treat a significant proportion of childhood BCP-ALLs.

MYC-an emerging player in mitochondrial diseases.

The mitochondrion is a major hub of cellular metabolism and involved directly or indirectly in almost all biological processes of the cell. In mitochondrial diseases, compromised respiratory electron transfer and oxidative phosphorylation (OXPHOS) lead to compensatory rewiring of metabolism with resemblance to the Warburg-like metabolic state of cancer cells. The transcription factor MYC (or c-MYC) is a major regulator of metabolic rewiring in cancer, stimulating glycolysis, nucleotide biosynthesis, and glutamine utilization, which are known or predicted to be affected also in mitochondrial diseases. Albeit not widely acknowledged thus far, several cell and mouse models of mitochondrial disease show upregulation of MYC and/or its typical transcriptional signatures. Moreover, gene expression and metabolite-level changes associated with mitochondrial integrated stress response (mt-ISR) show remarkable overlap with those of MYC overexpression. In addition to being a metabolic regulator, MYC promotes cellular proliferation and modifies the cell cycle kinetics and, especially at high expression levels, promotes replication stress and genomic instability, and sensitizes cells to apoptosis. Because cell proliferation requires energy and doubling of the cellular biomass, replicating cells should be particularly sensitive to defective OXPHOS. On the other hand, OXPHOS-defective replicating cells are predicted to be especially vulnerable to high levels of MYC as it facilitates evasion of metabolic checkpoints and accelerates cell cycle progression. Indeed, a few recent studies demonstrate cell cycle defects and nuclear DNA damage in OXPHOS deficiency. Here, we give an overview of key mitochondria-dependent metabolic pathways known to be regulated by MYC, review the current literature on MYC expression in mitochondrial diseases, and speculate how its upregulation may be triggered by OXPHOS deficiency and what implications this has for the pathogenesis of these diseases.

Abstract PR006: PMR-116, a novel inhibitor of ribosome biogenesis with antitumor activity in preclinical models of prostate cancer

Abstract Advanced prostate cancer is characterised by mutations and amplifications of genes involved in regulating protein synthesis. PTEN-loss stimulates activity of the mTOR pathway, while amplification of MYC leads to increased ribosome biogenesis and elevated mRNA translation rate. Our previous work has demonstrated the efficacy of co-targeting ribosome biogenesis, via inhibition of RNA Pol I activity, and 4E-BP1 phosphorylation to suppress prostate cancer growth in vivo in GEMM of PCa and in patient-derived xenografts(1-3). In a collaboration with Pimera Inc., we investigated the efficacy of their new lead RNA Pol I inhibitor PMR-116 in models of prostate cancer. PMR-116 is well tolerated in vivo in mice and can be given at 300mg/kg weekly. Using the Hi-MYC mouse model of PCa we show that dosing 6-month-old mice once weekly for 4 weeks can decrease the incidence of invasive lesions by up to 85% compared to vehicle control while reverting glands to patterns of low grade intraepithelial neoplasia. PMR-116 rapidly inhibits proliferation in the Hi-MYC model with a 50% decrease in Ki67 observed 12 hours after oral administration. Conversely, PMR-116 showed minimal anti-tumour efficacy in the PTEN-null model of PCa suggesting that elevated MYC signalling may be required for optimal response. To further validate our promising GEMM results in more clinically relevant human-derived models, we used patient-derived xenografts lines we established from multidrug-resistant, metastatic PCa(4). PMR-116 treatment decreased tumour volume in all PDX tested including complete response in a line in which tumour volume decreased by ~90% compared to baseline. We believe this new RNA Pol I inhibitor shows promising results in a wide range of preclinical models of androgen receptor dependent and independent PCa and may exert higher efficacy in tumours expressing high levels of MYC. PMR-116 is currently in Phase I dose escalation trial in patient with solid tumours (ACTRN12620001146987).

When Just One Phosphate Is One Too Many: The Multifaceted Interplay between Myc and Kinases.

Myc transcription factors are key regulators of many cellular processes, with Myc target genes crucially implicated in the management of cell proliferation and stem pluripotency, energy metabolism, protein synthesis, angiogenesis, DNA damage response, and apoptosis. Given the wide involvement of Myc in cellular dynamics, it is not surprising that its overexpression is frequently associated with cancer. Noteworthy, in cancer cells where high Myc levels are maintained, the overexpression of Myc-associated kinases is often observed and required to foster tumour cells' proliferation. A mutual interplay exists between Myc and kinases: the latter, which are Myc transcriptional targets, phosphorylate Myc, allowing its transcriptional activity, highlighting a clear regulatory loop. At the protein level, Myc activity and turnover is also tightly regulated by kinases, with a finely tuned balance between translation and rapid protein degradation. In this perspective, we focus on the cross-regulation of Myc and its associated protein kinases underlying similar and redundant mechanisms of regulation at different levels, from transcriptional to post-translational events. Furthermore, a review of the indirect effects of known kinase inhibitors on Myc provides an opportunity to identify alternative and combined therapeutic approaches for cancer treatment.

Abstract IA002: Inherited DNA repair defects and premature aging

Abstract Fanconi anemia (FA), a DNA repair disorder, is the most frequently inherited bone marrow failure (BMF) syndrome. Patients with FA suffer from early childhood onset of BMF, developmental abnormalities, and heightened susceptibility to solid tumors. FA patients also have a strong predisposition to myelo- dysplastic syndrome (MDS) and acute myeloid leukemia (AML). FA is caused by biallelic mutations in one of 23 FANC genes, whose protein products cooperate in the FA/BRCA DNA repair pathway and regulate cellular resistance to DNA cross-linking agents. Because of their underlying DNA repair defect, FA cells exhibit chromosomal instability and hypersensitivity to genotoxic DNA cross-linking agents, such as mitomycin C (MMC). FA bone marrow (BM) HSPCs are also hypersensitive to oxidative stress and inflammatory cytokines. FA patients and FA cells exhibit many features of Premature Aging. FA patients develop BMF because of HSPC exhaustion. Progressive age-related attrition is observed in CD34+ cell content in FA patients. Additionally, FA patients and FA mice exhibit HSPC functional defects. BMF in FA results from accumulation of DNA damage in HSPCs caused by endogenous cross-linking agents or physiological stress. In response to genotoxic stress, FA HSPCs hyperactivate growth-suppressive pathways, such as the p53 pathway (Ceccaldi et al, Cell Stem Cell, 2012) and the transforming growth factor (TGF-b) pathway (Zhang et al, Cell Stem Cell, 2016), further contributing to BMF. The molecular pathways in FA HSPCs leading to BMF and MDS/AML remain unknown. Although primary HSPCs from BM of FA patients are a useful model system, studying these cells is challenging because of their heterogeneity and low numbers. Sub-populations of HSPCs with heterogeneous transcriptional profiles may co-exist in the BM of FA patients. These subpopulations may include (1) stressed HSPCs sustaining hematopoiesis, (2) HSPCs committed to apoptosis resulting from accumulation of unrepaired DNA damage, and (3) premalignant/malignant cells that eventually lead to clinically detectable MDS or AML. Recently, in order to identify determinants of BMF, we performed single-cell transcriptome profiling of primary HSPCs from FA patients (Rodriguez et al, Cell Stem Cell, 2020). In addition to overexpression of p53 and TGF-b pathway genes, we identified high levels of MYC expression. We correspondingly observed coexistence of distinct HSPC subpopulations expressing high levels of TP53 or MYC in FA bone marrow (BM). MYC-high HSPCs showed significant downregulation of cell adhesion genes, consistent with enhanced egress of FA HSPCs from bone marrow to peripheral blood. We speculate that MYC overexpression impairs HSPC function in FA patients and contributes to exhaustion in FA bone marrow. In my seminar, I will describe the cellular and clinical manifestations of senescence and premature aging in FA patients. Specifically, FA patients exhibit the three major causes of cellular senescence including 1) replicative senescence 2) oncogene-induced senescence (OIS) and 3) stress-induced senescence. Citation Format: Alan D. D'Andrea. Inherited DNA repair defects and premature aging [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr IA002.

Apoptosis inhibition restrains primary malignant traits in different Drosophila cancer models.

Tumor cells exploit multiple mechanisms to evade apoptosis, hence the strategies aimed at reactivating cell death in cancer. However, recent studies are revealing that dying cells play remarkable pro-oncogenic roles. Among the mechanisms promoting cell death, cell competition, elicited by disparities in MYC activity in confronting cells, plays the primary role of assuring tissue robustness during development from Drosophila to mammals: cells with high MYC levels (winners) overproliferate while killing suboptimal neighbors (losers), whose death is essential to process completion. This mechanism is coopted by tumor cells in cancer initiation, where host cells succumb to high-MYC-expressing precancerous neighbors. Also in this case, inhibition of cell death restrains aberrant cell competition and rescues tissue structure. Inhibition of apoptosis may thus emerge as a good strategy to counteract cancer progression in competitive contexts; of note, we recently found a positive correlation between cell death amount at the tumor/stroma interface and MYC levels in human cancers. Here we used Drosophila to investigate the functional role of competition-dependent apoptosis in advanced cancers, observing dramatic changes in mass dimensions and composition following a boost in cell competition, rescued by apoptosis inhibition. This suggests the role of competition-dependent apoptosis be not confined to the early stages of tumorigenesis. We also show that apoptosis inhibition, beside restricting cancer mass, is sufficient to rescue tissue architecture and counteract cell migration in various cancer contexts, suggesting that a strong activation of the apoptotic pathways intensifies cancer burden by affecting distinct phenotypic traits at different stages of the disease.

Targeting MYC-driven lymphoma: lessons learned and future directions.

MYC plays a central role in tumorigenesis by orchestrating cell proliferation, growth and survival, among other transformation mechanisms. In particular, MYC has often been associated with lymphomagenesis. In fact, MYC overexpressing lymphomas such as high-grade B-cell lymphoma (HGBL) and double expressor diffuse large B-cell lymphomas (DLBCL), are considered addicted to MYC. In such a context, MYC targeting therapies are of special interest, as MYC withdrawal is expected to result in tumor regression. However, whether high MYC levels are always predictive of increased sensitivity to these approaches is not clear yet. Even though no MYC inhibitor has received regulatory approval to date, substantial efforts have been made to investigate avenues to render MYC a druggable target. Here, we summarize the different classes of molecules currently under development, which mostly target MYC indirectly in aggressive B-cell lymphomas, paying special attention to subtypes with MYC/BCL2 or BCL6 translocations or overexpression.

MYC and MET cooperatively drive hepatocellular carcinoma with distinct molecular traits and vulnerabilities

Enhanced activation of the transcription factor MYC and of the receptor tyrosine kinase MET are among the events frequently occurring in hepatocellular carcinoma (HCC). Both genes individually act as drivers of liver cancer initiation and progression. However, their concomitant alteration in HCC has not been explored, nor functionally documented. Here, we analysed databases of five independent human HCC cohorts and found a subset of patients with high levels of MYC and MET (MYChigh/METhigh) characterised by poor prognosis. This clinical observation drove us to explore the functionality of MYC and MET co-occurrence in vivo, combining hydrodynamic tail vein injection for MYC expression in the R26stopMet genetic setting, in which wild-type MET levels are enhanced following the genetic deletion of a stop cassette. Results showed that increased MYC and MET expression in hepatocytes is sufficient to induce liver tumorigenesis even in the absence of pre-existing injuries associated with a chronic disease state. Intriguingly, ectopic MYC in MET tumours increases expression of the Mki67 proliferation marker, and switches them into loss of Afp, Spp1, Gpc3, Epcam accompanied by an increase in Hgma1, Vim, and Hep-Par1 levels. We additionally found a switch in the expression of specific immune checkpoints, with an increase in the Ctla-4 and Lag3 lymphocyte co-inhibitory responses, and in the Icosl co-stimulatory responses of tumour cells. We provide in vitro evidence on the vulnerability of some human HCC cell lines to combined MYC and MET targeting, which are otherwise resistant to single inhibition. Mechanistically, combined blockage of MYC and MET converts a partial cytostatic effect, triggered by individual blockage of MYC or MET, into a cytotoxic effect. Together, these findings highlight a subgroup of HCC characterised by MYChigh/METhigh, and document functional cooperativity between MYC and MET in liver tumorigenesis. Thus, the MYC-R26Met model is a relevant setting for HCC biology, patient classification and treatment.

The Flavonoid Brusatol Induces Apoptosis in Aggressive Lymphoma Cells and Exhibits Synergistic Effect with Venetoclax

The most common lymphoid malignancies in adults are aggressive lymphomas, including Burkitt lymphoma and diffuse large B-cell lymphoma (DLBCL). Despite the available chemoimmunotherapy R-CHOP, one third of DLBCL patients still have primary refractory disease or relapse, and the incidence of these lymphomas continuously increases. Together, there is the great need for further development of new treatment strategies for aggressive lymphoma. One step forward seems to be Venetoclax (an FDA-approved Bcl-2 inhibitor), which showed an overall response rate of 44% in a phase 1 trial. Here, we evaluated a flavonoid - Brusatol, which showed anti-tumour activity in various preclinical cancer models, to improve anti-lymphoma therapies. We investigated the potential of Brusatol in the treatment of aggressive lymphoma cells as a single agent or in combination with other drugs, including Venetoclax. Seven cell lines representing different types of lymphoma (BL2, Raji (Burkitt lymphoma), SuDHL4, Karpas422 (GCB-DLBCL), RI-1, U2932 (ABC-DLBCL) and Jurkat (T-cell leukemia)) were treated with increasing concentrations of Brusatol (up to 10µM) for 72h to determine the IC50 values. Based on these results, 50nM and 250nM Brusatol were selected for further experiments. Cells were treated with Brusatol for 24h and 48h and apoptosis induction (Annexin V staining, Caspase-3- and PARP-cleavage) and changes in cell cycle (PI staining) were assessed. Moreover, samples from untreated and Brusatol-treated cells were collected for Western blot analysis. To investigate potential effects of Brusatol on protein synthesis in lymphoma cells, four cell lines (BL2, SuDHL4, RI-1 and U2932) were treated for 4h, and then the nascent protein synthesis was evaluated by detection of an incorporated methionine analogue using click-chemistry. Furthermore, co-treatment of Brusatol with inhibitors of Bcl-2 (Venetoclax), Bcl-XL (A-1331852), Mcl-1 (S63845), BTK (Ibrutinib), PI3K (Idealisib), Myc (EN4, JQ1), respectively, was performed in the same four cell lines and Annexin V levels were measured after 24h. Finally, the effect of combining Brusatol with Venetoclax was determined using the Bliss independence model for SuDHL4 and U2932 after 24h treatment via TMRE staining. In all investigated cell lines, Brusatol caused cell growth inhibition in a concentration-dependent manner. However, based on the results of the apoptosis assays, they could be grouped into cell lines more (BL2, Jurkat, Raji, SuDHL4) and less (Karpas422, RI-1, U2932) sensitive to Brusatol. Moreover, cell cycle analysis revealed an increase of cells in the sub-G1 phase in the more sensitive cell lines, indicating the induction of cell death. Western blot analysis of more sensitive cell lines showed decreased levels of the prosurvival proteins Bcl-2, Bcl-XL and Mcl-1. Additionally, we detected reduced p53 and Myc protein expression in cell lines more sensitive to Brusatol after 24h treatment. Notably, the protein expression profile of untreated cells indicates that cell lines with higher Myc levels are more sensitive to Brusatol treatment. Furthermore, mRNA expression analysis revealed that the reduction of affected proteins mainly occurred on protein level. Therefore, we investigated the effect of Brusatol on protein biosynthesis and observed protein translation inhibition in all tested cell lines. Interestingly, increased induction of apoptosis was apparent in all cell lines upon co-treatment with Brusatol and Bcl-XL inhibitor, in three of the four cell lines treated with the combination of Brusatol and Bcl-2 inhibitor, and in two cell lines when Brusatol was combined with Mcl-1 inhibitor. In contrast, combining Brusatol had no beneficial effect in combination with all other drugs tested. Finally, analysis of the Bliss independent model showed that the combination of Brusatol and Venetoclax synergistically enhanced the killing of lymphoma cells. Our data indicate that Brusatol efficiently induces cell death in aggressive lymphoma cells by reducing the expression of prosurvival proteins. Interestingly, cells with higher Myc levels were especially sensitive to Brusatol. Additionally, the combination of Brusatol with Venetoclax results in enhanced induction of apoptosis. Thus, our study suggests that Brusatol, alone or in combination with Venetoclax, represents a very interesting agent for further development of novel anti-lymphoma therapies.

Relocalization of PU.1 Underlies the On- and Off-Tumor Effects of SWI/SNF Blockade in AML

SWI/SNF ATP-dependent chromatin remodeling activity has been identified as a strong dependency of acute myeloid leukemia (AML). In AML cell lines, genetic knockdown or chemical inhibition of the SWI/SNF ATPases SMARCA4 (BRG1) or SMARCA2 (BRM) by the selective small-molecule inhibitor BRM014 reduces proliferation and induces leukemic differentiation. These effects arise because SWI/SNF inhibition reduces DNA accessibility at binding sites of PU.1, a factor essential for driving high levels of MYC in leukemic cells. However, given that both SWI/SNF and PU.1 are essential in hematopoietic development, important questions regarding the use of SWI/SNF inhibitors against AML remain. First, the chromatin features that influence SWI/SNF-dependent PU.1 regulation within healthy hematologic and leukemic cells have not been established. Furthermore, the ability of SWI/SNF inhibitors to target leukemic cells within the bone marrow has remained uncertain, and the effects of these drugs in an immunocompetent setting have not been reported, despite the essential functions of SWI/SNF and PU.1 in normal hematopoiesis. Finally, the sensitivity of primary human AML samples and normal hematopoietic cells has also not been established. As a result, many questions regarding the nature of the therapeutic window for SWI/SNF inhibition against AML have remained unresolved. Here we evaluate SWI/SNF inhibition using BRM014 in clinically relevant models and examine the consequences of the deregulation of SWI/SNF-dependent PU.1 sites. We demonstrate that inhibition of SWI/SNF shifts PU.1 occupancy from enhancers to promoters in both leukemic and healthy hematopoietic cells. In AML, the shift of PU.1 away from enhancers to the promoters of differentiation-related genes results in loss of MYC expression and differentiation of leukemic cells. In non-leukemic mice, SWI/SNF inhibition suppresses PU.1-dependent B cell development and bone marrow-derived monocyte populations, reflecting off-tumor effects caused by SWI/SNF-dependent PU.1 relocalization. These effects influence peripheral immune cells, as well lineage-committed hematopoietic progenitors within the bone marrow. Moreover, we observe profound therapeutic response to SWI/SNF inhibition in an immunocompetent AML mouse model (Figure 1). In vivo, BRM014 induces differentiation of peripheral leukemic blasts and reduces the leukemic stem cell burden in bone marrow but also induces lymphocytopenia due to its off-tumor effects on PU.1. Nevertheless, the robust regression of leukemic burden seen in vivo over a short two-week treatment period provides a compelling justification for continued study of SWI/SNF inhibitors in the treatment of AML. Further, response of primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) and AML specimens highlight that normal hematopoiesis is affected at concentrations similar to those needed for therapeutic response in AML (Figure 2). Hence, hematopoietic and immune-related side effects, particularly at higher doses and over the duration of treatment, will be important considerations for clinical use of SWI/SNF inhibitors against AML and other tumors. Overall, our results reveal a variable therapeutic window for SWI/SNF blockade in AML and highlight important off-tumor effects of such therapies in immunocompetent settings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting the MYC interaction network in B-cell lymphoma via histone deacetylase 6 inhibition

Overexpression of MYC is a genuine cancer driver in lymphomas and related to poor prognosis. However, therapeutic targeting of the transcription factor MYC remains challenging. Here, we show that inhibition of the histone deacetylase 6 (HDAC6) using the HDAC6 inhibitor Marbostat-100 (M-100) reduces oncogenic MYC levels and prevents lymphomagenesis in a mouse model of MYC-induced aggressive B-cell lymphoma. M-100 specifically alters protein-protein interactions by switching the acetylation state of HDAC6 substrates, such as tubulin. Tubulin facilitates nuclear import of MYC, and MYC-dependent B-cell lymphoma cells rely on continuous import of MYC due to its high turn-over. Acetylation of tubulin impairs this mechanism and enables proteasomal degradation of MYC. M-100 targets almost exclusively B-cell lymphoma cells with high levels of MYC whereas non-tumor cells are not affected. M-100 induces massive apoptosis in human and murine MYC-overexpressing B-cell lymphoma cells. We identified the heat-shock protein DNAJA3 as an interactor of tubulin in an acetylation-dependent manner and overexpression of DNAJA3 resulted in a pronounced degradation of MYC. We propose a mechanism by which DNAJA3 associates with hyperacetylated tubulin in the cytoplasm to control MYC turnover. Taken together, our data demonstrate a beneficial role of HDAC6 inhibition in MYC-dependent B-cell lymphoma.