MYC mRNA Expression Research Articles

Unbalanced MYC break-apart FISH patterns indicate the presence of a MYC rearrangement in HGBCL-DH-BCL2

AbstractFluorescence in situ hybridization (FISH) using break-apart probes is recommended for identifying high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2). Unbalanced MYC break-apart patterns, in which the red or green signal is lost, are commonly reported as an equivocal result by clinical laboratories. In a cohort of 297 HGBCL-DH-BCL2, 13% of tumors had unbalanced MYC break-apart patterns with loss of red (LR; 2%) or loss of green (LG; 11%) signal. To determine the significance of these patterns, MYC rearrangements were characterized by sequencing in 130 HGBCL-DH-BCL2, including 3 LR and 14 LG tumors. A MYC rearrangement was identified for 71% of tumors with LR or LG patterns, with the majority involving immunoglobulin loci or other recurrent MYC rearrangement partners. The architecture of these rearrangements consistently preserved the rearranged MYC allele, with the MYC gene predicted to be on the derivative chromosome containing the signal that is still present in nearly all cases. MYC protein expression, MYC messenger RNA expression, and the proportion of tumors expressing the dark-zone signature was not significantly different between balanced and unbalanced groups. These results support a recommendation that unbalanced MYC break-apart FISH patterns be reported as positive for MYC rearrangement in the context of diagnosing HGBCL-DH-BCL2.

A single, maximal dose of celecoxib, ibuprofen, or flurbiprofen does not reduce the muscle signalling response to plyometric exercise in young healthy adults.

Non-steroidal anti-inflammatory drugs (NSAIDs) possess analgesic and anti-inflammatory properties by inhibiting cyclooxygenase (COX) enzymes. Conflicting evidence exists on whether NSAIDs influence signaling related to muscle adaptations and exercise with some research finding a reduction in muscle protein synthesis signaling via the AKT-mTOR pathway, changes in satellite cell signaling, reductions in muscle protein degradation, and reductions in cell proliferation. In this study, we determined if a single maximal dose of flurbiprofen (FLU), celecoxib (CEL), ibuprofen (IBU), or a placebo (PLA) affects the short-term muscle signaling responses to plyometric exercise. This was a block randomized, double-masked, crossover design, where 12 participants performed four plyometric exercise bouts consisting of 10 sets of 10 plyometric jumps at 40% 1RM. Two hours before exercise, participants consumed a single dose of celecoxib (CEL 200mg), IBU (800mg), FLU (100mg) or PLA with food. Muscle biopsy samples were collected before and 3-h after exercise from the vastus lateralis. Data were analyzed using a repeated measures (RM) ANOVA, ANOVA, or a Friedman test. Significance was considered at p < 0.05. We found no treatment effects on the mRNA expression of PTSG1, PTSG2, MYC, TBP, RPLOP, MYOD1, Pax7, MYOG, Atrogin-1, or MURF1 (all, p > 0.05). We also found no treatment effects on AKT-mTOR signaling or MAPK signaling measured through the phosphorylation status of mTORS2441, mTORS2448, RPS6 235/236, RPS 240/244, 4EBP1, ERK1/2, p38 T180/182 normalized to their respective total abundance (all, p > 0.05). However, we did find a significant difference between MNK1 T197/202 in PLA compared to FLU (p < .05). A single, maximal dose of IBU, CEL, or FLU taken prior to exercise did not affect the signaling of muscle protein synthesis, protein degradation, or ribosome biogenesis three hours after a plyometric training bout.

SNHG15-mediated feedback loop interplays with HNRNPA1/SLC7A11/GPX4 pathway to promote gastric cancer progression.

Dysregulation of long noncoding RNA (lncRNA) expression plays a pivotal role in the initiation and progression of gastric cancer (GC). However, the regulation of lncRNA SNHG15 in GC has not been well studied. Mechanisms for ferroptosis by SNHG15 have not been revealed. Here, we aimed to explore SNHG15-mediated biological functions and underlying molecular mechanisms in GC. The novel SNHG15 was identified by analyzing RNA-sequencing (RNA-seq) data of GC tissues from our cohort and TCGA dataset, and further validated by qRT-PCR in GC cells and tissues. Gain- and loss-of-function assays were performed to examine the role of SNHG15 on GC both invitro and invivo. SNHG15 was highly expressed in GC. The enhanced SNHG15 was positively correlated with malignant stage and poor prognosis in GC patients. Gain- and loss-of-function studies showed that SNHG15 was required to affect GC cell growth, migration and invasion both invitro and invivo. Mechanistically, the oncogenic transcription factors E2F1 and MYC could bind to the SNHG15 promoter and enhance its expression. Meanwhile, SNHG15 increased E2F1 and MYC mRNA expression by sponging miR-24-3p. Notably, SNHG15 could also enhance the stability of SLC7A11 in the cytoplasm by competitively binding HNRNPA1. In addition, SNHG15 inhibited ferroptosis through an HNRNPA1-dependent regulation of SLC7A11/GPX4 axis. Our results support a novel model in which E2F1- and MYC-activated SNHG15 regulates ferroptosis via an HNRNPA1-dependent modulation of the SLC7A11/GPX4 axis, which serves as the critical effectors in GC progression, and provides a new therapeutic direction in the treatment of GC.

The Novel CDK9/CDK4/6/PI3K Triple Inhibitor LCI139 for the Treatment of MYC-Driven Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an aggressive B cell malignancy with characteristic t(11;14)(q13;q32) translocation juxtaposing IGH and CCND1 gene loci. This chromosomal rearrangement leads to Cyclin D1 overexpression and cell cycle dysregulation at the G1/S phase transition. MCL is generally diagnosed at advanced stages and follows an aggressive course of disease. Although chemotherapy-based combination regimens are initially effective, most patients relapse with a median survival of only 2.5-5 years. While novel targeted therapeutics have shown promising activity in relapsed/refractory patients, inevitable relapse associated with progressively declining efficacy and increasing resistance to single agent targeted therapy is a major concern. To overcome this barrier, we designed in-silico based a novel multitarget small molecule inhibitor LCI139 which simultaneously blocks three oncogenic targets: cyclin-dependent kinase 9 (CDK9), CDK4/6, and phosphatidylinositol-3 kinase (PI3K). LCI139 is a potent CDK9/CDK4/6/PI3K inhibitor with IC 50s of 0.000039µM (CDK9), 0.0015µM/0.0036µM (CDK4/6) 0.070µM (p110α), 0.461µM (p110γ), and 0.214 (p110δ), as determined by cell free assay alpha screens. Dose-dependent screening of LCI139 against a panel of MCL cell lines revealed robust cytotoxic effects with maximal IC 50 of 86.67 nM in JeKo-1 cells, 64.8 nM in Mino cells, and 202.5 nM in Rec-1 cells. Compared with clinical single agent CDK9 inhibitor AZD4573, LCI139 is less toxic to normal human stromal cells (HS-5), whereas both LCI139 and AZD4573 have no significant cytotoxic effect on normal human foreskin fibroblast cells (Hs68). LCI139 treatment at various concentrations for 24hrs induced significant apoptotic response in two MCL cell lines, JeKo-1 (p&amp;lt;0.001 DMSO vs LCI139 100 nM, p&amp;lt;0.0001 DMSO vs LCI139 200 nM) and Mino (p&amp;lt;0.05 DMSO vs LCI139 100 nM, p&amp;lt;0.0001 DMSO vs LCI139 200 nM) as determined by Annexin V/7-AAD apoptosis staining. Similarly, LCI139-induced apoptotic cell death was further confirmed by increasing cl-PARP protein levels compared to control as assessed by Western blot analysis. LCI139 induced robust G2/M cell cycle arrest in JeKo-1 cells and G1 arrest in Mino cells. The mechanism of action of pan-CDK inhibitors has been associated with downregulation of the anti-apoptotic protein MCL-1 through inhibition of transcriptional CDKs. Our results show that short-term exposure of LCI139 (1hr and 3hr) significantly suppressed MCL-1 and MYC mRNA expression in JeKo-1 (p&amp;lt;0.00001 DMSO vs LCI139) and Mino cells (p&amp;lt;0.00001 DMSO vs LCI139). LCI139 suppressed pRpb1 (Ser2), MCL-1 and cMYC protein expression in IgM-stimulated JeKo-1 and Mino cells in a dose-dependent manner. Treatment with LCI139 had no significant effect on pAKT (Ser473) in Jeko-1 cells, whereas pAKT (Ser473) was completely abolished in Mino cells at 500nM of LCI139. Mechanistically, our findings also demonstrate that LCI139 decreased MCL-1 and cMYC protein stability and reduced half-life as accessed by cycloheximide chase assay. This decrease in cMYC stability is associated with decreased of cMYC phosphorylation at Ser62. Adaptive kinome remodeling via drug-induced transcriptional reprogramming is a known contributor to ibrutinib resistant (IR) MCL. RNA-sequencing analysis of JeKo-1 vs Jeko-1 IR cells revealed a total of 3468 differentially expressed genes (FC &amp;gt; 1.5 and FDR &amp;lt; 0.05) where 110 kinases display altered expression in JeKo-1 IR cells. Finally, we show that LCI139 overcomes chronic ibrutinib resistance by decreasing viability in JeKO-1 IR and Mino IR cells with an IC 50 of 79 nM and 89 nM, respectively. Therefore, the multitarget small molecule inhibitor LCI139 has superior potency against IR MCL cell lines and overcomes ibrutinib-resistance at nanomolar doses.

Impact of curcumin on ferroptosis-related genes in colorectal cancer: Insights from in-silico and in-vitro studies.

Colorectal cancer (CRC)is responsible for a significant number of cancer-related fatalities worldwide. Researchers are investigating the therapeutic potential of ferroptosis, a type of iron-dependent controlled cell death, in the context of CRC. Curcumin, a natural compound found in turmeric, exhibits anticancer properties. This study explores the effects of curcumin on genes related to ferroptosis (FRGs)in CRC. To gather CRC data, we used the Gene Expression Profiling Interactive Analysis (GEPIA)and Gene Expression Omnibus (GEO)databases, while FRGs were obtained from the FerrDb database and PubMed. We identified 739 CRC differentially expressed genes (DEGs)in CRC and discovered 39 genes that were common genes between FRGs and CRC DEGs. The DEGs related to ferroptosis were enriched with various biological processes and molecular functions, including the regulation of signal transduction and glucose metabolism. Using the Drug Gene Interaction Database (DGIdb),we predicted drugs targeting CRC-DEGsand identified 17 potential drug targets. Additionally, we identified eight essential proteins related to ferroptosis in CRC, including MYC, IL1B, and SLC1A5. Survival analysis revealed that alterations in gene expression of CDC25A, DDR2, FABP4, IL1B, SNCA, and TFAM were associated with prognosis in CRC patients. In SW480 human CRC cells, treatment with curcumin decreased the expression of MYC, IL1B, and EZH2 mRNA, while simultaneously increasing the expression of SLCA5 and CAV1. The findings of this study suggest that curcumin could regulate FRGs in CRC and have the potential to be utilized as a therapeutic agent for treating CRC.

Refining Diagnostic Subtypes of Peripheral T-Cell Lymphoma Using a Multiparameter Approach

Peripheral T-cell lymphoma (PTCL) encompasses a diverse group of post-thymic lymphomas, with ~40% of cases not further classifiable and designated as PTCL-not otherwise specified (PTCL-NOS). Two molecular prognostic subtypes of PTCL-NOS, PTCL-TBX21 and PTCL-GATA3, were identified through gene expression profiling (GEP). A subset of PTCL-NOS with T-follicular helper (TFH) differentiation was subsequently categorized as nodal PTCL with TFH phenotype (PTCL-TFH), also known in current classifications as nodal PTCL-TFH, NOS, or TFH lymphoma, NOS. However, the boundary between PTCL-NOS and PTCL-TFH remains poorly defined. To refine these subtypes, 101 PTCL-NOS with centralized pathology review and extensive immunophenotyping by immunohistochemistry (IHC) using 22 antibodies, digital GEP (nCounter, NanoString Inc) (n= 70), and RNA sequencing (n= 73) were included in this study. IHC was used to identify PTCL-TFH cases (those with strong expression of more than two TFH markers); the remaining patients were classified as PTCL-GATA3 and PTCL-TBX21 using pre-defined GEP signatures. Cases were reclassified into PTCL-NOS (n= 63) and PTCL-TFH (n= 38), with PTCL-NOS subclassified into PTCL-GATA3 (n= 22; 34%) and PTCL-TBX21 (n= 41; 66%) and showing significant differences in OS (p&amp;lt;0.001). PTCL-GATA3 was characterized by medium to large transformed cells (average= 90%, 70-100%) and had minimal tumor microenvironment (TME) (TME-poor: 100%). In contrast, PTCL-TBX21 was more heterogeneous, associated with pleomorphic cells in a polymorphous background (TME-rich: 78%), including Lennert lymphoma-like cases (22%). mRNA and CIBERSORT analysis substantiated the findings and identified a subset enriched in cytotoxic features in the PTCL-TBX21 subtype. IHC indicated that PTCL-GATA3 cases were CD4+/CD8- (83%) or CD4-/CD8- (17%) and lacked expression of CD8 or cytotoxic markers compared to PTCL-TBX21 (p&amp;lt;0.01). PTCL-GATA3 showed significantly higher expression of LEF1 (average= 80%), Ki67 (80%), and MYC (25%) than PTCL-TBX21 (25%, 30%, &amp;lt;5%, respectively, p&amp;lt;0.01). mRNA and protein expression of these biomarkers showed a significant positive correlation (r=0.5, p&amp;lt;0.001), and expectedly higher mRNA expression of LEF1 and MYC was observed in the PTCL-GATA3 versus PTCL-TBX21 (p&amp;lt;0.05). Strong expression of CD30 (&amp;gt;50% of cells) was only seen in PTCL-GATA3 cases. EBER positivity, found only in rare background cells, was not significantly different (18% of PTCL-GATA3 vs. 12% of PTCL-TBX21). Within PTCL-TBX21, we identified cytotoxic and non-cytotoxic subsets with divergent morphological, phenotypic, and clinical findings. The cytotoxic PTCL-TBX21 exhibited an activated cytotoxic phenotype (23/25, 96%), denoted by TIA1 and granzyme-B and/or perforin expression. Extranodal involvement and single-cell apoptosis were observed in 17% and 45% of the cytotoxic cases, respectively, but absent in non-cytotoxic PTCL-TBX21 cases. A trend towards higher Ki67 expression (average= 40% vs. 20%, p= 0.08) was seen in the cytotoxic subgroup. In contrast, the non-cytotoxic PTCL-TBX21 was associated with a CD4+/CD8- phenotype and higher ICOS expression (average= 30%) and CCR4 (60%) compared to the cytotoxic PTCL-TBX21 (p= 0.001). PTCL-TFH cases showed a CD4+/CD8- phenotype (90%) and rarely a CD4-/CD8- phenotype (10%). While a subset of PTCL-TFH cases had AITL-like features such as numerous clear cells and/or prominent vasculature (31% of cases), which were not seen in the other subgroups, the remaining cases exhibited morphology that was indistinguishable from other PTLC-NOS cases including sheets of transformed cells or pleomorphic cells in a polymorphous background. Morphologically, PTCL-TFH cases, like PTCL-TBX21 were associated with a rich TME (TME-rich: 75%). CIBERSORT analysis showed enrichment of plasma cells (p&amp;lt;0.01) in PTCL-TFH, compared to PTCL-TBX21, and validated by morphology (30% of cases vs. 5%). Expression of one TFH marker was frequent in some PTCL-NOS cases (PTCL-GATA3= 41% of cases, PTCL-TBX21-non cytotoxic= 47%, PTCL-TBX21-cytotoxic= 5%). Conclusion: Our comprehensive evaluation underscores the importance of integrating morphology, immunophenotyping, and GEP in achieving an accurate diagnosis, potentially leading to more tailored treatment strategies for PTCL. Correlation with pending whole-exome sequencing studies will be provided at the time of presentation.

IGF2BP1 Stabilizes and Inhibits Degradation of Pro-Oncogenic Transcripts in ETV6-RUNX1 Positive B-Cell Acute Lymphoblastic Leukemia

Insulin like growth factor 2 mRNA binding protein 1(IGF2BP1) belongs to a family of oncofetal proteins expressed in embryonic life followed by re-emergence during malignant transformation. It is an RNA binding protein known to be dysregulated and overexpressed in multiple malignancies. We have demonstrated its specific overexpression in ETV6-RUNX1 translocated B-cell Acute Lymphoblastic Leukemia (B-ALL). RNA immunoprecipitation using anti-IGF2BP1 antibody, followed by sequencing (RIP-Seq) in Reh cell line (ETV6-RUNX1 positive) identified multiple oncogenic transcripts belonging to the non-canonical NF-κB and PI3K signaling pathways. Overexpression of genes from these pathways (IL6ST, GADD45A, NFAT5, MDM2, CDK6, FGFR1) was also observed in our B-ALL patient samples specifically in the ETV6-RUNX1 positive patients (Total n=111; ETV6-RUNX1 positive: 39) in comparison to MACS sorted, peripheral blood CD19 positive B-cells from healthy volunteers as well as in comparison to non ETV6-RUNX1 translocated B-Acute lymphoblastic leukemia patients. This was also corroborated by public B-ALL gene expression data from the TARGET database in the cBioportal. However, knockout of IGF2BP1 in Reh cell line using CRISPR-Cas9 technology led to significant downregulation of only some of these targets. This implied that the expression of some of the mRNA targets binding to IGF2BP1 was not influenced by its knockout. To better elucidate the role played by IGF2BP1 in the stability of its binding mRNA targets, we decided to study the rate of degradation of target transcripts. mRNA stability was assessed to quantify the rate of degradation of target mRNA after inhibition of transcription using actinomycin. Since actinomycin causes global transcriptional block, we first quantified the effect of actinomycin on the expression of multiple (n=7) housekeeping genes against each other, and selected 3 ( HGPRT, B2M and PPIA) which showed least change after actinomycin treatment. These 3 housekeeping genes were used for further quantification of target transcripts. Dual specificity phosphatase 1( DUSP1) a transcript known to be rapidly degraded, was used to validate the transcriptional inhibition by actinomycin. We quantified the rate of degradation of target transcripts in Reh cells using BTYNB, a small molecule inhibitor of IGF2BP1. BTYNB binds to IGF2BP1 and prevents binding to target transcripts which leads to their destabilization and reduced half-life. We used a combination of BTYNB and actinomycin to study rate of degradation after IGF2BP1 inhibition. Effect of IGF2BP1 on the stability of three target mRNAs involved in non-canonical NF-κB signalling pathway was quantified by comparing their rate of degradation in BTYNB treated cells against the DMSO (vehicle) treated cells. Rate of degradation was measured by actinomycin treatment of both these groups followed by hourly RNA isolation and qPCR over 12 hours. MYC is a known mRNA binding target of IGF2BP1. BTYNB was also shown to decrease the expression of MYC mRNA by accelerating its degradation. Simultaneously there was an increase in the expression of MYC pre-mRNA indicating a possible for IGF2BP1 in splicing of MYC RNA. In the NF-κB signalling pathway, we had previously identified that NFAT5 expression was not altered significantly after IGF2BP1 knockout even though it binds to IGF2BP1. BTYNB followed by actinomycin treatment demonstrated a dramatic increase in the rate of degradation of NFAT5 implying that IGF2BP1 prevents the degradation of NFAT5 transcript and directly influences its stability. Interestingly, the rates of degradation of other targets like IL6ST and GADD45 were not altered. These findings suggest a heterogeneous influence of IGF2BP1 on the stability of its target transcripts. Some of the transcripts seem to be directly stabilised by IGF2BP1 while others may be influenced indirectly. Taken together, these findings suggest a role of IGF2BP1 in multiple phases of the RNA synthesis and degradation warranting further studies to elucidate role of IGF2BP1 in each of these steps. IGF2BP1 appears to have a critical role in preventing the degradation and stabilizing MYC and NFAT5 transcripts.

Lipid metabolism reprogramming roles in mantel cell lymphoma growth and survival

Introduction: Metabolic reprogramming is a hallmark of cancer progression. However, there are few studies in mantle cell lymphoma (MCL). Protein arginine methyltransferase 5 (PRMT5), catalyzes monomethylation and symmetric demethylation of arginine residues on histone and nonhistone proteins and participates in tumor progression. This study aimed to investigate the mechanism of PRMT5-induced lipid reprogramming in MCL. Method: Lymph node, bone marrow biopsy, peripheral blood specimens and MCL cell lines were utilized as models. Liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS)-based analysis was performed to measure the steady-state level of metabolites. GSE93291 dataset and immunohistochemistry (IHC) staining were utilized to determine the relationship between expression levels of PRMT5 and patient outcome. CRISPR/Cas9 editing was employed to engineer the loss-of-function models of PRMT5 and other key genes involved in lipid metabolism. Result: Non-targeted LC-MS/MS analysis showed that lipid metabolites were rich in MCL cell lines (Figure 1A and 1B). Through systematic analysis of the relationship between lipid metabolism-related genes and MCL prognosis in GSE93291, PRMT5, SREBP1, SRENP2, FASN and MYC were found to be associated with the overall survival (OS) of MCL patients (Figure 1F–1J), which suggested that lipid metabolism reprogramming may be involved in the development of MCL. IHC staining of PRMT5 in 105 MCL tissues confirmed that elevated PRMT5 expression was significantly associated with poor progression free survival (PFS) (P = 0.039) and OS (P < 0.001) in MCL patients (Figure 1D and 1E). Multivariate Cox regression analysis showed that high PRMT5 expression was an independent prognostic indicator of OS (P = 0.003). Gene transcriptome sequencing showed downregulation of the PRMT5 caused the reduced expression of SREBP1/SREBP2 and FASN and change the expression of lipid metabolite (Figure 1K). PRMT5, SREBP1/SREBP2 and FASN depletion significantly impaired outgrowth and increased apoptosis of MCL cells. In primary tumor samples, MYC protein levels were significantly correlated with PRMT5 protein levels (P < 0.001). PRMT5 knockout blocked MYC mRNA and protein expression, suggesting a requisite role of PRMT5 in MYC transcription regulation. Further molecular biological methods were used to verify that PRMT5 could participate in lipid metabolism reprogramming through MYC by changing the expression levels of SREBP1/SREBP2 and FASN (Figure 1L). Keywords: Aggressive B-cell non-Hodgkin lymphoma, Diagnostic and Prognostic Biomarkers, Metabolism No conflicts of interests pertinent to the abstract.

DDDR-30. UNRAVELING THE NOVEL ANTI-TUMOR MECHANISM OF ST-401, A BRAIN PENETRANT MICROTUBULE TARGETING AGENT

Abstract Microtubule targeting agents (MTAs) are indispensable medicines to treat a wide range of cancers. While evidence suggests that glioblastoma (GBM) is sensitive to disruptions in microtubule (MT) functions; most MTAs do not reach the brain. To address this limitation, we developed a brain-penetrant MTA, ST-401, that kills patient-derived GBM in culture at nanomolar concentrations, binds to colchicine sites through a unique interaction mode, gently and reversibly inhibits MT assembly and kills cancer cells through a novel anti-tumor mechanism as determined by analysis of its activity in the NCI 60 cancer cell panel compared to the Approved Oncology Agents library (Horne et al. (2020) Neuro-Onc Adv. 3(1): vdaa165).We used flow cytometry to confirm that nocodazole (10-100 nM) treatment of HCT116 cells triggered apoptosis, necrosis, and accumulation of autophagosomes, as well as increase in p-P53. By sharp contrast, ST-401 did not trigger these responses. Live cell imaging showed that NOC preferentially kills HCT116 cells in mitosis whereas ST-401 preferentially kills these cells in interphase. ScRNAseq analysis of NOC and ST-401 treatment of HCT-116 cells showed that ST-401 down-regulates MYC mRNA expression, suggesting that ST-401 might affect MYC function. MYC protein levels are regulated by multiple mechanisms, including phosphorylation at residue Thr58 that promotes its ubiquitination and proteasome degradation. We discovered that ST-401 increases MYC phosphorylation on Thr58 and down-regulates MYC protein expression in both HCT116 and the GBM cell line, U251.Our result suggests that the novel anti-tumor mechanism of ST-401 might involve cell death in interphase and reduced MYC expression. Funded by NIH (CA244213 to NS and NS106924 to NS).

Prediction and verification of the key ingredients and molecular targets of Guizhi Fuling capsule against tumour metastasis and resistance

PurposeThe well-known traditional Chinese formula Guizhi Fuling capsule (GFC) has been reported to reverse ovarian cancer drug resistance. Extrachromosomal DNA (ecDNA) plays an important role in tumour metastasis and resistance. The purpose of this study was to investigate the potential mechanisms by which GFC blocks tumour metastasis and reverses drug resistance by targeting ecDNA.MethodsCNKI and PubMed were used to obtain pharmacokinetic research data on GFC in rats, and the bioactive ingredients detected in rat serum or plasma were collected. Network databases were used to screen the abnormally expressed genes in ecDNA, tumour metastasis genes, resistance genes, and the active ingredient targets of GFC. The KOBAS3.0 database was used to enrich the KEGG pathways and GO functions; the STRING platform was used to construct the core protein interaction network; and the molecular docking online tool SwissDock was used to analyse the binding activity of the core targets and the active ingredients. RT-qPCR, Western blotting and laser confocal microscopy were used to verify the effect of the sera containing GFC on ecDNA, mRNA and protein expression of key targets.ResultsTwenty-three bioactive ingredients of GFC were retrieved from PubMed and CNKI. Nine shared targets were simultaneously involved in abnormal genes in ecDNA, tumour metastasis and resistance and the active ingredient targets of GFC. GO functional analysis indicated that the cotargets involved cell proliferation, apoptotic regulation, nuclear functions, etc. The potential pathways involved in the reversal of tumour metastasis and drug resistance of GFC were the PI3K-Akt signalling, cancer, and platinum drug resistance pathways. Three shared proteins targeting ecDNA (AKT1, EGFR and MYC) stand out from the top 20 PPI targets, and all of the bioactive ingredients of GFC have strong binding affinity to the three proteins. The active ingredients can reduce the expression of MYC, EGFR and AKT1 mRNA and protein and the amount of ecDNA in drug-resistant OC cells.ConclusionsGFC targeting ecDNA to reverse tumour metastasis and drug resistance has the characteristics of multiple ingredients, multiple targets, and multiple pathways, which provides a new perspective for the development of new drugs targeting ecDNA to benefit tumour treatment.

Efficient Identification of the MYC Regulator with the Use of the CRISPR Library and Context-Matched Database Screenings.

MYC is a major oncogene that plays an important role in cell proliferation in human cancers. Therefore, the mechanism behind MYC regulation is a viable therapeutic target for the treatment of cancer. Comprehensive and efficient screening of MYC regulators is needed, and we had previously established a promoter screening system using fluorescent proteins and the CRISPR library. For the efficient identification of candidate genes, a database was used, for which mRNA expression was correlated with MYC using datasets featuring “Similar” and “Not exactly similar” contexts. INTS14 and ERI2 were identified using datasets featuring the “Similar” context group, and INTS14 and ERI2 were capable of enhancing MYC promoter activity. In further database analysis of human cancers, a higher expression of MYC mRNA was observed in the INTS14 mRNA high-expressing prostate and liver cancers. The knockdown of INTS14 in prostate cell lines resulted in decreased MYC mRNA and protein expression and also induced G0/1 arrest. This study confirmed that CRISPR screening combined with context-matched database screening is effective in identifying genes that regulate the MYC promoter. This method can be applied to other genes and is expected to be useful in identifying the regulators of other proto-oncogenes.

Abstract 3267: ARID1A mutated bladder cancer cells are sensitized to BET protein inhibition with OTX-015

Abstract Background: Bladder cancer (BC) is a common and deadly disease. Inactivating ARID1A mutations occur in up to 30% of metastatic BC tumors, making it the most commonly mutated epigenetic gene and the 4th most commonly mutated gene overall in BC. ARID1A mutations are associated with decreased response to therapy and poor prognosis; thus, there is a need to develop therapies specifically targeting ARID1A mutant tumors. Using the UNC EpiG Diamond compound library, a set of well-annotated small molecule probes targeting chromatin regulatory proteins, we determined that inhibitors of bromodomain and extraterminal (BET) proteins potently inhibit the viability of BC cells. Here, we tested the hypothesis that ARID1Amut cells are particularly sensitized to the pan-BET inhibitor OTX-015. Methods: AR1D1A-competent (ARID1AWT) 5637 and ARID1A-mutated (ARID1Amut) HT1197 cells were treated with eight ascending concentrations of OTX-015 (0.1 nM-100 µM), and cell viability was measured using CellTiter-Glo™ after 72-120-hour incubations. IC50 values were calculated using a four-parameter non-linear regression model. Gene expression of BET inhibitor target genes, MYC, ARID1B, and RAD51, were evaluated by RT-PCR after a 48-hour incubation, normalized to an SDHA housekeeper and compared to a 0.1% DMSO control. CellTiter-Glo™ and RT-PCR experiments were conducted in technical and biologic triplicates. Western blotting evaluated OTX-015 effects on c-MYC, BAF250B (encoded by ARID1B), and RAD51 protein expression after 72-hour incubation. Results: OTX-015 was 8-times more potent in ARID1Amut HT1197 cells at 120 h than in ARID1AWT 5637 cells (IC50: 0.12 μM vs. 1.0 μM). OTX-015 treatment (1 µM) significantly reduced ARID1B and RAD51 mRNA expression versus 0.1% DMSO control (83% and 86% reduction, respectively, both P&amp;lt;0.0001). At the same concentration, OTX-015 did not significantly reduce MYC mRNA expression HT1197 cells (25% reduction, P=0.31), but did significantly reduce MYC expression in 5637 cells (57% reduction, P&amp;lt;0.0001) at 48 h. When comparing HT1197 to 5637 cells, OTX-015 treatment (1 µM) caused a greater reduction in ARID1B mRNA expression (83% vs. 62% reduction, P=0.02) and RAD51 mRNA expression (86% vs. 57%, P=0.001) at 48 h. Finally, OTX-015 treatment (1 µM) also resulted in more dramatically reduced c-MYC, ARID1B and RAD51 protein expression in HT1197 cells at 72 h, when compared to 5637 cells. Conclusions: These preliminary results support future lines of inquiry into the molecular mechanisms that underlie sensitization of ARID1Amut cells to BET protein inhibition. Citation Format: Ryan M. Kemper, Heemaja Mewada, Jeffrey S. Damrauer, Brian Hardy, Stephen F. Frye, Kenneth H. Pearce, Jesse Raab, William Y. Kim, Daniel James Crona. ARID1A mutated bladder cancer cells are sensitized to BET protein inhibition with OTX-015 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3267.

P-307 Modulation of the MYC oncogene using programmable epigenetic mRNA therapeutics as a novel therapy for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a leading cause of cancer deaths worldwide with a significant unmet medical need. MYC over-expression is associated with aggressive disease in up to ∼70% of HCC. While MYC represents an attractive therapeutic target, it has historically been considered undruggable, due to a lack of a structured binding pocket and tightly regulated expression. The MYC gene and its regulatory elements are part of an insulated genomic domain (IGD), a chromatin looping region anchored by CTCF. At Omega, our approach is to epigenetically modulate gene expression pre-transcriptionally utilizing engineered, programmable, mRNA therapeutics called Omega Epigenomic Controllers (OECs), to target IGDs. The development candidate, OTX-2002 is an OEC targeted to the MYC IGD and its activity has been characterized in vitro through MYC mRNA expression, cell viability, and epigenetic changes and in vivo in HCC models by assessing changes in tumor volume, immunohistochemistry (IHC) to detect MYC expression and other biomarkers, and epigenetic alterations. Furthermore, non-human primates (NHPs) were dosed with OTX-2002 to evaluate effects on MYC mRNA expression translationally across species. Additionally, combinations of OTX-2002 with agents used to treat HCC (e.g. tyrosine kinase inhibitors sorafenib and lenvatinib) as well as anti-PD-(L)1 checkpoint inhibitor therapies were tested in vitro along with proteomics analysis (Somalogic) to identify potential synergizing agents. OTX-2002 was effective at decreasing MYC mRNA, protein and cell viability in HCC cells while sparing normal cells in vitro. In HCC cells, OTX-2002 median EC50 of inhibition is 2 weeks, providing durable MYC mRNA repression. IV delivery of OTX-2002 in vivo at doses of 3 and 6 mg/kg Q5D in a Hep3B subcutaneous model demonstrated significant tumor growth inhibition of 54% and 63%, respectively, with no decrease in body weight. OTX-2002 also showed activity in additional HCC subcutaneous and orthotopic xenograft models. IHC of OTX-2002-treated tumors showed significant down-regulation of MYC with reduced proliferation and increased apoptosis. OTX-2002-induced epigenetic changes were confirmed in extracted tumors. Serially-collected liver biopsies from NHPs treated with OTX-2002 exhibit significant downregulation of MYC mRNA expression 24 hrs (71.9% reduction) and 7 days (75.4% reduction) post-dosing compared to baseline. Combination of OTX-2002 with sorafenib or lenvatinib showed additivity and/or synergy in vitro and in vivo in HCC models. Furthermore, OTX-2002 can decrease surface PD-L1 expression in HCC cells, suggesting a potentially advantageous combination with checkpoint inhibitors. Proteomic analysis also revealed pathways whose inhibition synergizes with OTX-2002’s mechanism of action, including ERK1/2 signaling, NF-kB signaling, and autophagy. OECs enable pre-transcriptional modulation of the MYC oncogene through precise epigenomic programming of the IGD. Our pre-clinical data show activity both in mice and NHPs. Targeting MYC in this manner may represent a new and potentially effective approach to the treatment of HCC, either as a single agent or in combination with various approved or investigational therapeutics. Omega plans to submit an IND for OTX-2002 in HCC in 1H2022.

PKP1 and MYC create a feedforward loop linking transcription and translation in squamous cell lung cancer.

Plakophilin 1 (PKP1) is well-known as an important component of the desmosome, a cell structure specialized in spot-like cell-to-cell adhesion. Although desmosomes have generally been associated with tumor suppressor functions, we recently found that PKP1 is recurrently overexpressed in squamous cell lung cancer (SqCLC) to exert an oncogenic role by enhancing the translation of MYC (c-Myc), a major oncogene. In this study, we aim to further characterize the functional relationship between PKP1 and MYC. To determine the functional relationship between PKP1 and MYC, we performed correlation analyses between PKP1 and MYC mRNA expression levels, gain/loss of function models, chromatin immunoprecipitation (ChIP) and promoter mutagenesis followed by luciferase assays. We found a significant correlation between the mRNA levels of MYC and PKP1 in SqCLC primary tumor samples. In addition, we found that MYC is a direct transcription factor of PKP1 and binds to specific sequences within its promoter. In agreement with this, we found that MYC knockdown reduced PKP1 protein expression in different SqCLC models, which may explain the PKP1-MYC correlation that we found. Conversely, we found that PKP1 knockdown reduced MYC protein expression, while PKP1 overexpression enhanced MYC expression in these models. Based on these results, we propose a feedforward functional relationship in which PKP1 enhances MYC translation in conjunction with the translation initiation complex by binding to the 5'-UTR of MYC mRNA, whereas MYC promotes PKP1 transcription by binding to its promoter. These results suggest that PKP1 may serve as a therapeutic target for SqCLC.

IBtkα Activates the β-Catenin-Dependent Transcription of MYC through Ubiquitylation and Proteasomal Degradation of GSK3β in Cancerous B Cells.

The IBTK gene encodes the IBtkα protein that is a substrate receptor of E3 ubiquitin ligase, Cullin 3. We have previously reported the pro-tumorigenic activity of Ibtk in MYC-dependent B-lymphomagenesis observed in Eμ-myc transgenic mice. Here, we provide mechanistic evidence of the functional interplay between IBtkα and MYC. We show that IBtkα, albeit indirectly, activates the β-catenin-dependent transcription of the MYC gene. Of course, IBtkα associates with GSK3β and promotes its ubiquitylation, which is associated with proteasomal degradation. This event increases the protein level of β-catenin, a substrate of GSK3β, and results in the transcriptional activation of the MYC and CCND1 target genes of β-catenin, which are involved in the control of cell division and apoptosis. In particular, we found that in Burkitt’s lymphoma cells, IBtkα silencing triggered the downregulation of both MYC mRNA and protein expression, as well as a strong decrease of cell survival, mainly through the induction of apoptotic events, as assessed by using flow cytometry-based cell cycle and apoptosis analysis. Collectively, our results shed further light on the complex puzzle of IBtkα interactome and highlight IBtkα as a potential novel therapeutic target to be employed in the strategy for personalized therapy of B cell lymphoma.

CN470 is a BET/CBP/p300 multi-bromodomain inhibitor and has an anti-tumor activity against MLL-rearranged acute lymphoblastic leukemia

Acute lymphoblastic leukemia with chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene (MLL-r ALL) remains an incurable disease. Thus, development of a safe and effective therapeutic agent to treat this disease is crucial to address this unmet medical need. BRD4, a member of the bromodomain and extra-terminal domain (BET) protein family, and cyclic AMP response element binding protein binding protein (CBP) and p300, two paralogous histone acetyltransferases, are all considered cancer drug targets and simultaneous targeting of these proteins may have therapeutic advantages. Here, we demonstrate that a BET/CBP/p300 multi-bromodomain inhibitor, CN470, has anti-tumor activity against MLL-r ALL in vitro and in vivo. CN470, potently inhibited ligand binding to the bromodomains of BRD4, CBP, and p300 and suppressed the growth of MLL-r ALL cell lines and patient-derived cells with MLL rearrangements. CN470 suppressed mRNA and protein expression of MYC and induced apoptosis in MLL-r ALL cells, following a cell cycle arrest in the G1 phase. Moreover, CN470 reduced BRD4 binding to acetylated histone H3. The in vivo effects of CN470 were investigated using SEMLuc/GFP cells expressing luminescent markers in an orthotopic mouse model. Mice administered CN470 daily had prolonged survival compared to the vehicle group. Further, CN470 also showed anti-tumor effects against an MLL-r ALL patient-derived xenograft model. These findings suggest that inhibition of BET/CBP/p300 by the multi-bromodomain inhibitor, CN470, represents a promising therapeutic approach against MLL-r ALL.

Inhibition of FLT3-ITD and Melk By Small Molecule Inhibitor OTS167 in FLT3 mutant Acute Myeloid Leukemia (AML)

Background and Rationale: Maternal embryonic leucine zipper kinase (MELK) is a Ser/Thr protein kinase that is aberrantly expressed in many tumor types and demonstrated to be important in formation and maintenance of cancer stem cells. We have previously reported that MELK is also aberrantly expressed in AML cell lines and primary patient leukemia cells and is associated with a poor prognosis (Alachkar et. al., Oncotarget, 2014). MELK knockdown resulted in leukemia cell growth inhibition and apoptosis. Based on these preclinical insights, a small molecule inhibitor of MELK kinase activity, OTS167, (OncoTherapy Sciences, Tokyo, Japan) has been developed and is being tested in an early phase clinical trial in advanced myeloid malignancies at the University of Chicago (IRB#15-1373). Here, we report the activity of MELK signaling blockade in AML with FLT3 mutations, which are associated with drug resistance and poor clinical outcomes, and demonstrate synergy with known FLT3 kinase inhibitors.Methods: Both MELK and FLT3-ITD activity were targeted by treating the human AML cell lines MV4:11, MOLM-13, KOCL-48 and KG-1 which aberrantly express MELK as well as a FLT3-ITD, FLT3-TKD or FLT3-WT with the small molecule MELK inhibitor OTS167 alone or in combination with FLT3 kinase inhibitors Crenolanib or Gilteritinib.Results: OTS167 induced cell death of FLT3 mutant cell lines in a dose dependent manner and with IC50 similar to known FLT3 kinase inhibitors (OTS167 IC50 1.8-18.2nM; Crenolanib IC50 2.31-13.8nM; Gilteritinib IC50 4.9-36.3nM). Using isobologram analysis, OTS167 in combination with either Gilteritinib or Crenolanib induced synergistic cell death in FLT3 mutant leukemia cells at 48hrs (Figure 1A), but not in FLT3-WT leukemia cells. We found that OTS167 alone had the effect of inhibiting auto-phosphorylation of FLT3-ITD, as well as inhibiting MELK activity resulting in downregulation of MELK protein expression. Treatment of cells with 50nM OTS167 resulted in loss of FLT3-ITD (Y591) phosphorylation within 1hr, and total FLT3-ITD protein expression within 24 hours (Figure 1B). Loss of FLT3-ITD protein expression occurred in the presence of FLT3 mRNA and was not due to decreased stability of the protein, as treatment with OTS167 in the presence of cycloheximide did not decrease FLT3-ITD lifespan. Notably, the loss of FLT3-ITD protein expression observed with OTS167 treatment was not observed with the same concentration of FLT3 inhibitors Crenolanib or Gilteritinib. In vitro kinase assays using recombinant FLT3-ITD confirmed that OTS167 could act directly to inhibit FLT3-ITD kinase activity at an IC50 of 4.9nM, compared to a reported MELK IC50 of 0.11-1.1nM (Chung, et. al., Oncotarget, 2012). In MV4:11 cells, the constitutive activation of the MAPK, PI3K, and STAT5 signaling pathways by FLT3-ITD is inhibited by 50nM OTS167, similar to inhibition by 50nM FLT3 inhibitors Crenolanib and Gilteritinib (Figure 1C).In addition, treatment with either OTS167 or FLT3 inhibitor resulted in loss of MYC mRNA and protein expression at 3hr, suggesting that more effective downregulation of MYC expression by the combination may increase the susceptibility of FLT3 mutant AML to apoptosis. The inhibition of FLT3-ITD activation and expression after OTS167 treatment was not observed when MELK protein activity was downregulated using siRNA, suggesting the inhibitor may simultaneously target two independent signaling pathways active in cellular transformation. These results indicate that OTS167 may have a dual specificity in FLT3 mutant AML through targeting both aberrant MELK and FLT3 activity.Conclusion: In summary, the MELK inhibitor, OTS167, may be an attractive therapeutic agent for FLT3 mutant AML, with dual targeting resulting in decreased levels of FLT3-ITD protein and kinase activity. These data provide a rationale for testing dual MELK and FLT3 kinase inhibitors in patients with FLT3 mutant AML. [Display omitted] DisclosuresOdenike:CTI/Baxalta: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria. Stock:Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees.

HUWE1 Inhibition As a Therapeutic Strategy to Target MYC in MM

BackgroundThe use of proteasome inhibitors to disrupt the ubiquitin proteasome system (UPS) has provided a significant therapeutic advance and highlights the importance of this pathway in Multiple Myeloma (MM). There is increasing interest in developing therapeutic strategies to target enzymes that regulate the UPS, with the aim of gaining more specificity and reducing side-effects. We previously identified elevated expression of the E3 ligase HUWE1 in MM compared to normal cells and demonstrated that inhibition of HUWE1 with shRNA results in growth arrest of MM cell lines (Blood 2016, 128:240). HUWE1 is involved in the regulation of MYC and its expression is deregulated in a number of tumor types. The aim of this study was to elucidate the role and therapeutic potential of inhibiting HUWE1 in MM using small molecule inhibitors.MethodsInhibitors of HUWE1 (BI8622, BI8626) as described in Peter et al. (EMBO Mol Med 2014, 6:1525-1541) were purchased from Syngene. The effect of the inhibitors on MM cell lines (OPM-2, KMS-18, JJN3, MM.1S, MM.1R, ANBL6.WT, ANBL6.BR), patient cells and normal bone marrow (NBM) from healthy donors was assessed using CellTitre-glo and WST1 assays. Cells were analysed for cell cycle status (propidium iodide and EdU staining), apoptosis and β-galactosidase activity. Snapshot Proteomics (AVM Biomed) was performed on HUWE1 knockdown cells to identify novel ubiquitinated substrates of HUWE1. Signalling pathways and substrates of HUWE1 were analysed using UbiQapture-Q (Enzo Life Sciences), Western blotting and RQ-PCR. Calcusyn software was used to calculate a combination index (CI) to assess synergistic anti-MM activity.ResultsUbiQapture assays demonstrated that BI8622 and BI8626 blocked auto-ubiquitination of HUWE1 in a concentration-dependent manner, with an associated decrease in the ubiquitination of MIZ1 (a validated HUWE1 substrate). BI8622 and BI8626 significantly reduced the growth of MM cell lines, including those resistant to dexamethasone (MM.1R) and bortezomib (ANBL6.BR), and patient-derived MM cells (p ≤ 0.01; IC50 range 9-20 µM) without significantly affecting NBM, suggesting a favourable therapeutic index. Furthermore, both compounds inhibit the proliferation of MM cell lines when co-cultured with MM patient-derived bone marrow stromal cells. HUWE1 inhibition induced cell cycle arrest, associated with a reduction of cells undergoing DNA synthesis (p = 0.005), cellular senescence demonstrated by β-galatosidase staining and increased p16 protein expression. HUWE1 inhibition in MM cell lines resulted in a significant decrease in MYC mRNA and protein expression (p < 0.001), accompanied by downregulation of multiple MYC-dependent target genes (CCND2, HSPE1, ODC1, CCNA2, CDC25A; p ≤ 0.02). Proteomic profiling along with ingenuity pathway analysis revealed novel putative substrates of HUWE1 involved in the regulation of MYC. Finally, the combination of HUWE1 inhibitors with proteasome inhibitors (carfilzomib and bortezomib) and dexamethasone results in synergistic anti-MM activity (CI range 0.57 - 1).ConclusionsMYC is frequently dysregulated in MM and represents an attractive therapeutic target. Here we demonstrate that inhibitors of the E3 ligase HUWE1 lead to growth arrest of MM cell lines, associated with decreased expression of MYC and MYC-dependent target genes. While BI8622 and BI8626 have previously been reported to repress MYC activity in models of colorectal cancer, we demonstrate for the first time that in MM this is accompanied with a decrease in MYC expression. Studies are ongoing to elucidate the molecular mechanism of MYC downregulation following HUWE1 inhibition both in vitro and in vivo. In summary, this study demonstrates that HUWE1 is important in the growth and survival of MM cells and presents a novel therapeutic strategy for the inhibition of MYC function in MM. DisclosuresNo relevant conflicts of interest to declare.

Dual Inhibition of Aurora Kinase and BRD4 Leads to Improved Anti-Tumor Activity in Myc-Overexpressing Lymphoma Cells

Background:Myc has proven extremely difficult to target therapeutically. Therefore, we hypothesized that optimal inhibition of multiple key pathways involved in Myc expression could overcome this long-standing problem. We identified aurora A kinase (AURKA) and bromodomain protein (BRD)4 as therapeutic targets to counteract Myc deregulation based on strong evidence that these pathways are essential for tumor maintenance in Myc-driven malignancies.Methods: Cytotoxicity assays using MTS and trypan blue were used to compare levels of drug sensitivity in lymphoma cell lines with high and low Myc mRNA expression. Apoptosis and cell cycle assays were performed using Annexin V and Propidium Iodide staining. Cycloheximide chase followed by immunoblotting was used to examine Myc protein degradation kinetics, and real-time RT-PCR was used to examine Myc mRNA levels. Murine xenograft models were used to assess the efficacy and tolerability of single vs. combined inhibition.Results: Myc-overexpressing Burkitt lymphoma (Raji) cells were treated with increasing concentrations of small molecule inhibitors against AURKA (MLN-8237), BRD4 (I-BET-151), or both drugs for 48 to 72 hours and cell viability was evaluated. MLN-8237 and I-BET-151 inhibited cell growth individually with IC-50 of 10 nM, and 650 nM, respectively. As compared to treatment with the single agent alone, dual treatment induced more significant cell growth inhibition with IC-50 achieved at drug concentrations of < 5 nM and 500 nM for MLN-8237 and I-BET-151, respectively. The combination index (CI) values were less than 1 at various drug concentrations, indicating that different combinations were synergistic in terms of inhibitory effect on tumor cell viability. Superior activity of the dual inhibition was also noted in different Myc-overexpressing lymphoma cells. Combination treatments increased apoptosis and induced more pronounced cell cycle arrest compared to the single agent treatment alone.We then analyzed protein expression by Western blot. MLN-8237 reduced the expression of p-HisH3 and p-AURKA while increasing the expression of p-S6K. Treatment with I-BET-151 resulted in significant reduction of Myc total protein. Cycloheximide chase experiments demonstrated that inhibition of AURKA increased the rate of Myc protein degradation, while BRD4 inhibition had no effect on degradation kinetics. Conversely MLN-8237 did not decrease Myc transcription, while I-BET-151 significantly reduced the levels of Myc mRNA. The combination of the two drugs demonstrated additive/synergistic decreases of both Myc protein degradation and Myc transcription at higher drug doses.Lastly, athymic nude mice bearing Ramos lymphoma xenografts were treated with MLN-8237, I-BET-151, or with the combination of both agents (n = 8 per group). The mean tumor volumes in mice treated with negative control, I-BET-151, and MLN-8237 as single agents were 3480, 2320, and 671 mm3, respectively, at Day 28. Mice treated with MLN-8237/I-BET-151 combinations had mean tumor volume of 461 mm3 at Day 28. The survival rates for mice treated with negative control, I-BET-151, and MLN-8237 as single agents were 0%, 10%, and 70%, respectively, at Day 35 (Figure 1). Mice treated with the MLN-8237 and I-BET-151 combination demonstrated a 100% survival rate at Day 35.Conclusion: Our data demonstrated that Myc-overexpressing lymphoma can be successfully targeted by inhibition of Myc transcription and/or enhancement of Myc protein degradation. Specifically, MLN-8237, a small molecule inhibitor against AURKA, induced apoptosis of Myc-overexpressing tumor cells in vitro and showed the most promising anti-tumor activity in mice bearing Myc-overexpressing lymphoma, especially when combined with I-BET-151, a BRD4 inhibitor. [Display omitted] DisclosuresPark:Cornerstone Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead: Speakers Bureau; Seattle Genetics: Research Funding; Teva: Consultancy, Research Funding; Takeda: Research Funding. Ghosh:TG Therapeutics: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding, Speakers Bureau; Jassen: Consultancy, Honoraria, Research Funding; Kite Pharma: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Speakers Bureau; Seattle Genetics: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau. Sharpless:Unity Biotechnology: Other: NA; HealthSpan Diagnostics: Other: NA; Pfizer: Other: NA; G1 Therapeutics: Equity Ownership.

MTOR-C/EBPβ-c-Myc Axis Regulates the Behavior of Myeloid-Biased Hematopoietic Stem Cells Under Stress Conditions

In stress or regenerative conditions, hematopoietic stem cell (HSC) population drastically alters its behavior; they rapidly enter into cell cycle, differentiate to multipotent progenitors (MPPs) at the expense of self-renewal activity, and are reprogrammed toward predominantly myeloid-biased hematopoiesis. In these processes, essential roles of myeloid transcription factors in regenerating HSCs are anticipated. C/EBPβ regulates both proliferation and differentiation of granulocytic precursors in stress conditions. However, its functions in HSCs during stresses are largely unknown.In the past several years, we have been investigating the roles of C/EBPβ in HSCs under stress conditions, especially focusing on the expression pattern of C/EBPβ isoforms and their respective functions. Cebpb is a unique single exon gene, from which two long protein isoforms (LAP* and LAP) and one truncated isoform lacking N-terminal activating domain (LIP) are translated by different usage of initiating codons. To monitor the expression pattern of these isoforms in scarce cells, we have devised an intracellular double staining method for flow cytometric analysis using two distinct anti-C/EBPβ antibodies recognizing N- and C-terminus, respectively (57th ASH annual meeting, #3580).Here, we refined this method to reveal the characteristic expression pattern of C/EBPβ isoforms in HSCs in vivo in response to stresses. In regenerative conditions after either 5-FU treatment or transplantation, we detected remarkable LIP-dominant upregulation in LT-HSCs at early phases, which was followed by increase of LAP*/LAP at later phases. This stress-induced translational upregulation and alteration of C/EBPβ were significantly suppressed by short-term administration of rapamycin, a selective inhibitor of mTOR, suggesting its dependence on mTORC1 pathway at least in part.We further found that the stress-induced LIP-dominant upregulation of C/EBPβ was remarkable in LT-HSCs with higher surface CD150 expression (CD150high LT-HSCs), which possess myeloid-biased potential. Cell cycle analyses at early phases after 5-FU treatment, when LIP was predominantly expressed, revealed that CD150high HSCs were significantly less quiescent than CD150low HSCs but this difference was abolished in Cebpb-/- HSCs, suggesting that C/EBPβ specifically activated the cell cycle of CD150high “myeloid-biased” HSCs.We next examined the functions of C/EBPβ isoforms in HSCs in vivo . We transplanted WT BM cells retrovirally transduced with each C/EBPβ isoform into sublethally irradiated WT mice, and then analyzed the transduced cells in the recipients. LAP*- or LAP-transduced cells disappeared as early as 3 weeks after transplantation, probably due to their rapid myeloid differentiation. LIP-transduced HSCs were significantly less quiescent, exhibited more accelerated differentiation to MPPs, especially lymphoid-primed MPP4s, and produced more lymphoid-biased outputs. These results indicate that LIP induces cell cycle activation of HSCs and expansion of MPP pool, while antagonizing the other long isoforms that induce rapid myeloid commitment and premature exhaustion of HSCs.As a candidate for the downstream target of C/EBPβ, especially LIP, in HSCs, we focused on Myc (encoding c-Myc). LIP-transduced EML cells, a mouse HSC line, expressed 1.5-fold higher level of Myc mRNA than those transduced with control, and LIP-transduced HSCs in vivo expressed 2-fold higher than control. We next compared Myc mRNA expression between WT and Cebpb-/- HSCs at early phases of regeneration post 5-FU treatment and transplantation, when LIP was predominantly upregulated. Then, we found that significantly lower level of Myc transcripts was expressed in Cebpb-/- HSCs. When we performed a ChIP-qPCR analysis using C/EBPβ-transduced EML cells, remarkable enrichment of C/EBPβ was observed at the distal enhancer of Myc gene. These data suggest that both forced overexpression and physiological elevation of LIP positively regulate Myc transcription in HSCs likely through direct binding to Myc enhancer.In summary, our findings illustrate that stress-induced upregulation and alteration of C/EBPβ protein, mediated by mTORC1 and modulating Myc transcription, is significantly involved in stress-mode behavior of HSCs especially with myeloid bias. DisclosuresNo relevant conflicts of interest to declare.