c-Myc Protein Expression Research Articles

S104: RBPS DYSREGULATION CAUSE HYPER-NUCLEOLI AND RIBOSOME GAIN-OF-FUNCTION DRIVING BONE MARROW FAILURE

Background: Nucleoli and ribosome cross-talk regulates cell translation capacity. Its dysregulation and impairment drive ribosome and nucleolus stress (NS), related to the biological mechanism of cancer. Ribosomopathies are a group of diseases characterized by ribosome defects leading to complex syndromes that include bone marrow failure. hnRNP K is an RNA binding protein (RBP) in charge of processing nascent RNAs (nucleoli) into mature mRNAs (ribosome). Our research found a novel ribosome gain-of-function ribosomopathy phenotype by hyper-nucleoli generation due RBP hnRNP K dysregulation. Aims: We aim to elucidate how hnRNP K dysregulation impact on haematopoietic stem cell biology. Methods:Hnrnpk overexpression was established in MEFs using CRISPR/SAM (Konermann S. et al, Nature). Global protein synthesis was tested using a Click-iT OPP and proteasome function was evaluated by Proteasome 20S activity NS hallmarks were analyzed by confocal microscopy evaluating Ncl, NS sensor marker. To evoke NS in our cells, we used Actinomycin D insult. Cell cycle FACS analysis (DAPI) and senescence assays (β-galactosidase staining) were performed. Molecular mechanism underlying was elucidated by qRT-PCR and WB (p21, p16, c-Myc, and mTor). To study the impact of hnRNP K overexpression in vivo, we developed an inducible tamoxifen mouse model activated 30-60 days after birth (HnrnpkTg-Ubc-creERT2). Survival was evaluated by Kaplan-Meier, and phenotype described by symptoms, signs, CBC and bone marrow IHC panel (CD34, Gr1, B220, MPO). Results:Hnrnpk overexpressing cells led to an increment in protein and gene expression of Ncl, mTor and c-Myc (Figure 1A-B). Moreover, we found an increase in global protein synthesis (Figure 2C-D). Nevertheless, the elevation of hnRNP K inversely correlated with the proteasome function, which dropped significantly (Figure 1E). NS induction promoted higher hnRNP K expression. Additionally, hnRNP K overexpressing cells showed NS hallmarks associated with an increase of the number of nucleoli, and total area of the nucleoli and nucleus (Figure 1F-G). Cell cycle analysis confirmed an increment of arrested G2/M phase cells (Figure 1H-I), linked to an increment in p21 and p16 levels all leading towards a senescent cell phenotype (Figure 1J-L). HnrnpkTg-Ubc-creERT2 mice had widespread Hnrnpk overexpression (Figure 1M) and a reduction in lifespan (Figure 1N), mainly due to dysplastic and bone marrow failure phenotype, with dramatic reduction of CD34 and b-cells, leukopenia, anaemia and thrombocytopenia. (Figure 1O-Q). Image:Summary/Conclusion: The overexpression of hnRNP K drives to an increase in nucleoli activity, leading to ribosome biogenesis and higher global translation by the regulation of molecules involved in both systems: Ncl, c-Myc or mTor. Our work found that hnRNP K overexpression in vivo drives a bone marrow failure phenotype, promoting the exhaustion of haematopoietic stem cells by ribosome dysregulation that triggered cell senescence. Of note, this is the first time reported that a nucleoli/ribosome-gain-of-function induce bone marrow failure ribosomopathies phenotype. This work was financially supported by CRIS contra el Cancer Association (NGO) AES ISCIII (PI18/00295), ISCIII Miguel Servet (CP19/00140), Cancer Research UK [C355/A26819], FC AECC and AIRC under the Accelerator Award Program and National Cancer Institutes of Health Award (R01CA207204, SMP) Leukemia and Lymphoma Society (6577-19, SMP).

PB2141: CLINICOPATHOLOGICAL FEATURES AND OUTCOME OF CD5 POSITIVE DIFFUSE LARGE B CELL LYMPHOMA AND THE SIGNIFICANCE OF CD5 EXPRESSION STRENGTH: 10-YEAR SINGLE CENTER EXPERIENCE

Background: CD5 positive (CD5+) diffuse large B cell lymphoma (DLBCL) is a rare subtype of DLBCL encompassing 5-10% of patients (pts) with DLBCL, and it is characterized by poor outcome. Aims: To identify clinicopathological features of CD5+ DLBCL in a cohort of pts managed at Clinic for Hematology, Clinical Center of Serbia during a 10-year period (2012-2021). Methods: We retrospectively analyzed immunohistochemistry (IHC) results of biopted tumor tissue of 537 pts with DLBCL NOS. Survival analysis was performed using Kaplan-Meier method and log-rank test. Results: Out of a total of 537 pts, in 211 (39.3%) CD5 was not analyzed by IHC, while in the remaining 326 (60.7%) was. Among them, 27 (8.3%) were found to be positive for CD5, out of which 10 pts were designated at the pathology report as CD5 +/- (5 pts) or CD5 -/+ (5 pts), while the rest 17 pts were clearly CD5+. In CD5 positive cohort, 52% of pts were men and median age at diagnosis was 63 years. ECOG performance status (PS) was poor (≥2) in 8 (29.6%) pts. One third of pts had advanced stage disease (Ann Arbor III/IV), while the rest two thirds were limited stage (I and II). Eleven out of 26 pts (42.3%) presented with B symptoms, 3/26 (11.5%) expressed bulky disease, while two thirds presented with extranodal disease. More pts (13/23 or 56.5%) were of nonGCB subtype according to IHC, while 4 out of 11 pts (36.4%) analyzed for c-myc protein expression were double expressors. Median Ki-67 proliferative index was 80%. Almost half of pts (47.6%) had increased initial lactate dehydrogenase (LDH) activity, while median beta-2 microglobulin (b2m) level was 3.38 mg/L. Nine out of 26 pts (34.6%) were IPI>2, while the rest 17 pts were IPI 0-1. Vast majority (25/27, 92.6%) were treated with chemoimmunotherapy in the first line, most of them with anthracycline-based protocols, 1 patient received cyclophosphamide, vincristine, prednisone (COP), and 1 patient succumbed before treatment commencement. Among treated pts with known outcome after first line, 18 (72%) pts achieved complete remission, 4 (16%) achieved partial response, while 3 (12%) pts did not respond to treatment. Median progression-free survival (PFS) after first treatment line for all pts in CD5 positive cohort was 25 months (95%CI 13.76-36.24), while median overall survival (OS) was not reached. Age at diagnosis, presence of B symptoms, and extranodal disease did not significantly influenced PFS and OS. Patients with bulky disease, nonGCB subtype, and double expressors experienced significantly shorter PFS (p=0.03, P=0.043, and p=0.025, respectively), but these features did not impact OS. On the other hand, b2m levels >3.38 mg/L, LDH >1x upper limit normal, and IPI >2 were significantly associated with shorter OS (p=0.014, p=0.01, and p=0.015, respectively), while they were not significant regarding PFS. ECOG PS >1 was significantly associated with both poor PFS and OS (p=0.005 and p=0.002, respectively). Intrestingly, pts designated as CD5+ at pathology reports experienced median PFS of 20 months in contrast to 52 months in pts descripted as CD5+/- and CD5-/+ (p=0.021). However, the difference between these two subgroups regarding OS was not observed. Summary/Conclusion: In our CD5+ DLBCL cohort, bulky disease, nonGCB, and double expressors were associated with shorter PFS, higher b2m, LDH, and IPI correlated with shorter OS, while higher ECOG PS was associated with shorter both PFS and OS. Median PFS after 1st treatment line was around 2 years, while median OS was not reached. Strenght of CD5 expression was significantly associated with PFS. CD5 should be mandatory within IHC for DLBCL.

TRIM52 promotes proliferation, invasion, and migration of gastric cancer cells by regulating Wnt/β-catenin pathway

Purpose: This study aimed to reveal the role and mechanism of tripartite motif-containing 52 (TRIM52) in gastric cancer (GC) progression.Methods: The Cancer Genome Atlas (TCGA) database was utilized to analyze TRIM52 expression in GC samples and para-carcinoma tissue samples, and the results were confirmed by quantitative realtime polymerase chain reaction. Cell counting kit-8 and colony formation assays were used to evaluate cell viability. Wound healing assay was utilized to analyze cell migration, while Transwell assay was utilized to evaluate cell invasion. TRIM52, proliferating cell nuclear antigen, matrix metalloproteinase-2, Wnt5a, β-catenin, and c-Myc protein levels were measured by western blot.Results: TRIM52 was expressed more in GC tissue samples and cells compared to normal tissues and cells (p < 0.001). Overexpression of TRIM52 promoted growth, migration, and invasion of HGC-27 cells, and silencing inhibited growth, migration, and invasion of HGC-27 cells (p < 0.001). In addition, TRIM52 overexpression increased Wnt5a, β-catenin, and c-Myc protein expression, and silencing decreased Wnt5a, β-catenin, and c-Myc protein expression (p < 0.001 or p < 0.01), indicating that TRIM52 activates Wnt/β-catenin signaling pathway.Conclusion: These findings reveal that TRIM52 facilitates GC cell proliferation, migration and invasion, but activates Wnt/β-catenin signaling.

Alternative c-MYC mRNA Transcripts as an Additional Tool for c-Myc2 and c-MycS Production in BL60 Tumors.

While studying c-Myc protein expression in several Burkitt lymphoma cell lines and in lymph nodes from a mouse model bearing a translocated c-MYC gene from the human BL line IARC-BL60, we surprisingly discovered a complex electrophoretic profile. Indeed, the BL60 cell line carrying the t(8;22) c-MYC translocation exhibits a simple pattern, with a single c-Myc2 isoform. Analysis of the c-MYC transcripts expressed by tumor lymph nodes in the mouse λc-MYC (Avy/a) showed for the first time five transcripts that are associated with t(8;22) c-MYC translocation. The five transcripts were correlated with the production of c-Myc2 and c-MycS, and loss of c-Myc1. The contribution of these transcripts to the oncogenic activation of the t(8;22) c-MYC is discussed.

Abstract 4030: Efficacy of novel combination therapy against AML cells with mutant NPM1

Abstract Cytoplasmic dislocation and loss of nucleolar localization of the chaperone protein NPM1c occurs in 30% of AML following a frame shift C-terminal mutation in exon 12 of NPM1 (creating NPM1c), which contributes to differentiation arrest, growth and survival of AML stem progenitor cells. In present studies, we determined that knock out (KO) of mutant (mt) NPM1 (NPM1c) in the AML OCI-AML3, utilizing targeted gRNA (compared to control) and CRISPR-Cas9, induced growth arrest, morphologic differentiation and loss of viability of the KO cells over 5 to 9 days. This was associated with depletion of protein levels of NPM1c, c-Myc, and MEIS1, but upregulation of PU.1, RUNX1, CD11b, p21 and NOXA levels. ChIP-Seq analysis with H3K27Ac antibody 5d after KO demonstrated reduced peak density at the super-enhancers of MYC, MEIS1, and HOXA9. RNA-Seq analysis showed negative enrichment of HALLMARK gene sets of mRNA expression of MEIS1/HOXA9, MYC targets, ribosome/translation and cell cycle. Compared to OCI-AML3 with NPM1c, OCI-AML3 with NPM1 KO exhibited abrogation of the Menin inhibitor SNDX-50469-induced differentiation and apoptosis. Additionally, NPM1 KO also significantly reduced sensitivity of OCI-AML3 cells to apoptosis induced by targeted agents previously shown to demonstrate efficacy against AML cells with NPM1c, including KPT-330 (XPO1 inhibitor), homoharringtonine (protein translation inhibitor), and anti-AML chemotherapeutic agents, including daunorubicin, cytarabine and etoposide. However, NPM1 KO sensitized OCI-AML3 cells to ATRA (all trans retinoic acid)-induced differentiation and loss of viability, which was associated with marked induction of p21 and CD11b. Utilizing RNA-Seq signature of NPM1c KO and interrogating the LINCS1000-CMap data set of gene expression signatures, we determined that among the top expression mimickers were several pan-HDAC inhibitors and a WEE1 kinase inhibitor. Treatment with adavosertib (WEE1 inhibitor, MK-1775) or romidepsin (HDAC inhibitor) dose-dependently induced apoptosis in OCI-AML3 cells, as well as in 5 samples of patient-derived (PD) AML cells with NPM1c. Moreover, co-treatment with adavosertib and romidepsin synergistically induced lethality in OCI-AML3 and PD AML cells with NPM1c. This was associated with marked reduction in protein expressions of c-Myc, c-Myb, MEIS1 and CDK4/6, but upregulation of TP53 and p21 levels. These findings highlight that presence of NPM1c mechanistically regulates the sensitivity of AML cells to Menin inhibitor, ATRA and anti-AML chemotherapeutic agents. They also identify the novel non-chemotherapy combination of pan-HDAC inhibitor and WEE1 inhibitor for its potential synergistic activity against AML with NPM1c. Citation Format: Christopher P. Mill, Warren C. Fiskus, John A. Davis, Courtney D. DiNardo, Christine Birdwell, Qi Jin, Koichi Takahashi, Joseph D. Khoury, Kapil N. Bhalla. Efficacy of novel combination therapy against AML cells with mutant NPM1 [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 4030.

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<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<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.

Abstract 6298: The novel long noncoding RNA lncNeur promotes neuroblastoma by up-regulating AURKA and Myc expression

Abstract Background: Neuroblastoma is the most common solid tumor in early childhood, and accounts for approximately 15% of childhood cancer death. We have recently analyzed RNA sequencing data from 493 human neuroblastoma tissues, and identified the long noncoding RNA lincNeur as one of the transcripts, high expression of which most considerably correlated with poor prognosis in both MYCN oncogene-amplified and MYCN non-amplified neuroblastoma patients. Aims: To determine the tumorigenic role of lincNeur in neuroblastoma, and to identify the mechanism of action through which lincNeur exerts oncogenic function, also to screen for small molecule compound inhibitors of lincNeur. Methods and results: We performed RNA-binding protein pull-down assay and RNA-immunoprecipitation assay. RALY was identified and validated as one of the binding proteins of lincNeur. We also identified down-stream targets of lincNeur via genome-wide differential gene expression analysis. E2F1 and AURKA were identified as among the transcripts significantly down-regulated after lincNeur knockdown. RT-PCR and western blot analysis showed that lincNeur knockdown reduced E2F1 and AURKA mRNA and protein expression, and reduced N-Myc and c-Myc protein but not mRNA expression. In addition, lincNeur or RALY knockdown reduced, while over-expression of lincNeur or RALY promoted, neuroblastoma cell proliferation. We also demonstrated lincNeur knockdown significantly suppressed RALY protein binding to E2F1 and AURKA mRNAs. Mouse xenograft model experiment showed that lincNeur knockdown suppressed neuroblastoma tumor progression in vivo. Importantly, we have confirmed the binding site between lincNeur and RALY, and are screening small molecule compound libraries for inhibitors of lincNeur and RALY interaction for neuroblastoma therapy. Conclusions: The novel long noncoding RNA lincNeur promotes neuroblastoma by interacting with RALY to up-regulate E2F1 and AURKA mRNA and protein, as well as N-Myc and c-Myc protein expression. Small molecule compound inhibitors of lincNeur and RALY interaction is a promising therapeutic approach. Citation Format: Jing Wu, Pei Yan Liu, Belamy Cheung, Glenn Marshall, Marcel Dinger, Tao Liu. The novel long noncoding RNA lncNeur promotes neuroblastoma by up-regulating AURKA and Myc expression [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 6298.

Abstract 1128: MTORC1/2 and HDAC1/2 inhibition promote tumor response through inhibition of MYC

Abstract Background: The identification of treatment strategies targeting PIK3CA mutant colorectal cancer (CRC) are of great clinical interest. Previous work from our lab has identified MTORC1/2 and HDAC1/2 inhibition with copanlisib (cop; PI3K/MTOR) and romidepsin (romi; HDAC1/2) as a potential therapeutic strategy. We hypothesized that changes in c-MYC protein levels and c-MYC target gene (CTG) alterations might be a potential mechanism of action for this combination. Methods: Known CTGs were identified and their expression levels were examined in PIK3CA mutant vs WT CRCs using the cBioPortal Colorectal Adenocarcinoma (TCGA, PanCancer Atlas) dataset. Murine derived cancer organoids (MDCO) were generated from adenocarcinomas of Apc and Pik3ca mutant transgenic mice (F1 (FVB x B6) Apcfl/+ Pik3caH1047R/+). MDCO results were corroborated using the human 2D isogenic cell lines SW48 and SW48PIK3CA-H1047R (SW48PK) and RAS/RAF WT patient derived cancer organoids (PDCO) generated from CRC patient samples under approved IRB protocols. Immunoblots (IB) were used to assess c-MYC levels after treatment with cop, romi, and the combination across all models. RNA sequencing was conducted on PDCOs and SW48PK cells and changes in 16 CTGs were examined. An aggregate score was created for each treatment group where a statistically significantly altered CTG with log fold change ≥1.5 added one point and ≤-1.5 subtracted one point. All others were scored 0. Results: c-MYC target genes were assessed for differential expression in PIK3CA mutant CRC vs PIK3CA WT CRC with only 1/16 genes decreased in PIK3CA mutant CRC (GADD45A: log ratio -0.21, q=0.01). Next, MDCOs treated with the combination showed a decrease in total c-MYC levels in the combination therapy. These results were corroborated in two human 2D CRC cell lines, SW48 and SW48PK and a panel of PDCOs. Interestingly, c-MYC levels decreased in both romi alone and the combination in both the SW48 and SW48PK cell lines after 24 hours of treatment. However, the extent to which c-MYC levels were decreased was not as substantial in the panel of PDCOs. Across all in vitro models a decrease in PI3K signaling, as illustrated by decreased pRPS6 and p4EBP1 in response to cop treatment and an increase in H3K27 acetylation in response to romi, was observed in the single agent and combination therapies. RNA sequencing demonstrated that cop, romi, and combo had a CTG score of 0, -4, and -7 respectively in the PIK3CA mutant PDCO. Similar results were seen in an additional RAS/RAF WT PDCO (0, 1, and -4, respectively) and SW48PK cells (0, -3, and -7, respectively). Conclusion: A potential mechanism by which cop and romi treatment promote tumor response is through a decrease in c-MYC protein levels and expression of downstream c-MYC target genes. This regimen deserves further mechanistic investigations in vivo. Citation Format: Rebecca A. DeStefanis, Autumn M. Olson, Alyssa K. DeZeeuw, Susan N. Payne, Cheri A. Pasch, Linda Clipson, Dustin A. Deming. MTORC1/2 and HDAC1/2 inhibition promote tumor response through inhibition of MYC [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 1128.

Abstract 1809: Engineered destabilized ARE on the 3UTR of MYC degrades oncogenic c-MYC transcript and protein and induces apoptosis in multiple cancer types

Abstract The c-MYC oncogene is overexpressed in more than 18 human cancers, including triple negative breast cancer (TNBC), prostate cancer, and osteosarcoma, yet there is no clinically approved drug that targets this major oncogenic driver. We discovered that the 3’UTR of c-MYC in many cancers is enriched with stabilizing poly U tract sequences that can be destabilized with our novel engineered stabilizing AU Rich Elements (ARE), leading to degradation of c-MYC. We hypothesize that c-MYC destabilizing constructs will degrade c-MYC transcript and proteins across a range of cancers that overexpress this oncogene. We used heterogeneous panels of TNBC, prostate cancer, and osteosarcoma cell lines that are reflective of the heterogeneity of human cancers. We introduced our c-MYC destabilizing construct and assessed cell viability. We then performed immunofluorescence and western blot for assessment of c-MYC protein expression. Vector constructs were used as controls. Introduction of destabilizing elements achieved the degradation of both c-MYC transcript and proteins within four days, as well as a subsequent loss in cell viability. We observed that treated cells disintegrated from their nucleus, leading to increased active caspase 3/7 and cell death. Taken together, we have developed a novel technology that reliably targets and degrades the c-MYC oncogene across a range of highly aggressive and difficult to treat cancers Citation Format: Chidiebere Awah, Fu Dong, Yana Glemaud, Fayola Levine, Daniel Weiser, Olorunseun Ogunwobi. Engineered destabilized ARE on the 3UTR of MYC degrades oncogenic c-MYC transcript and protein and induces apoptosis in multiple cancer types [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 1809.

Abstract 1728: Single cell RNA-seq analysis reveals a role of pro-inflammatory tumor-associated macrophages in driving prostate cancer progression

Abstract Genomic amplification and increased expression of C-MYC are found in high-grade intraepithelial neoplasm (HGPIN) and primary prostate adenocarcinoma. What role C-MYC plays in the initiation and progression of prostate cancer remains unclear. A genetically engineered mouse model with a high level of human C-MYC protein expression under a probasin promoter (Hi-Myc model) was developed that captures prostate cancer progression from hyperplasia at 6-week-old, to HGPIN at 3-6 month-old, and ultimately to invasive adenocarcinoma at 8-month-old. Applying single-cell RNA sequencing (scRNA-seq) technology, we have carefully studied the temporal transcriptional change in MYC-expressing tumor-initiating cells and the tumor-infiltrating immune cells to understand the cell-intrinsic and -extrinsic mechanisms by which C-MYC promotes the transition from HGPIN to invasive prostate cancer. We have identified a particular type of luminal cells, Prom1+ L1 cells, as the likely tumor initiating cells in the Hi-Myc model. The MYC-expressing L1 tumor initiating cells were more abundant and cycling more often in the invasive cancer at 8-month-old, compared to the pre-invasive stage. Interestingly, significant infiltration of novel M1-like tumor-associated macrophages (TAMs) correlated with the histologic tumor progression from HGPIN to invasive cancer. Targeting TAMs with anti-CSF1R antibody delayed prostate cancer progression and invasion, which appears to be independent of CD8 T-cells. Mechanistically, TAMs expressed a high level of IL-1b, which stimulated Hi-Myc prostate cell proliferation in an organoid co-culture system. More importantly, depleting IL-1b with a neutralizing antibody slowed prostate cancer progression in Hi-Myc mice. Lastly, we found that increased macrophage gene expression was associated with higher Gleason scores and worse disease-free survival in a TCGA clinical patient cohort. In summary, we have identified a molecular mechanism involving pro-inflammatory TAMs and IL-1b in driving early prostate cancer progression in a MYC-driven prostate cancer model, opening a potential therapeutic avenue to target TAMs and pro-inflammatory cytokine in preventing early prostate cancer progression. Citation Format: Jimmy L. Zhao, Joseph Chan, Max Land, Perianne Smith, Anuradha Gopalan, Michael Haffner, Dana Pe'er, Charles Sawyers. Single cell RNA-seq analysis reveals a role of pro-inflammatory tumor-associated macrophages in driving prostate cancer progression [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 1728.

A Novel Biomarker, FKBP10, for Poor Prognosis Prediction in Patients with Clear Cell Renal Cell Carcinoma.

Objective To screen genes associated with poor prognosis of clear cell renal cell carcinoma (CcRCC) from the public databases HPA (Human Protein Atlas), UALCAN, and GEPIA (Gene Expression Profiling Interactive Analysis) and to investigate the expression of FKBP10 in CcRCC and the effect on prognosis of the patients and the biological behavior of CcRCC cells. Methods The tumor tissues and adjacent noncancerous tissues of 42 patients with CcRCC diagnosed and treated in our hospital were collected, and the general information of the patients was recorded. FKBP10 expression in the tissues was determined by qRT-PCR and western blot, and its relationship with general information and prognosis of patients was analyzed. Knockdown or overexpression experiments were carried out with the human proximal tubule epithelial cell line HK-2 and CcRCC cell lines Caki-1, 786-O, ACHN, and A498 to verify the relationship between FKBP10 expression and cell proliferation and adhesion ability using MTT assay and fibronectin adhesion assay, respectively. Western blot was utilized to examine the protein expression level of c-Myc, cyclin D1, and Bcl-2 in the cells. Results FKBP10 was highly expressed in CcRCC tissues and cells and was correlated with poor prognosis. In addition, FKBP10 expression was positively correlated with CcRCC tumor size and staging and negatively correlated with tumor differentiation. Moreover, knockdown of FKBP10 significantly inhibited the proliferation of CcRCC cells, notably declined the protein expression of c-Myc, cyclin D1, and Bcl-2, and promoted cell adhesion. Conclusion FKBP10 is highly expressed in CcRCC tissues and cells and is associated with poor prognosis in patients. FKBP10 participated in the occurrence and development of CcRCC by promoting cell proliferation and inhibiting apoptosis and adhesion.

β-Escin reduces cancer progression in aggressive MDA-MB-231 cells by inhibiting glutamine metabolism through downregulation of c-myc oncogene

The c-myc oncogene, which causes glutamine dependence in triple negative breast cancers (TNBC), is also the target of one of the signaling pathways affected by β-Escin. We sought to determine how c-myc protein affects glutamine metabolism and the proteins, glutamine transporter alanine-serine-cysteine 2 (ASCT2) and glutaminase (GLS1), in β-Escin-treated MDA-MB-231 cells using glutamine uptake and western blot analysis. Cell viability, colony formation, migration and apoptosis were also evaluated in MDA-MB-231 cells in response to β-Escin treatment using MTS, colony forming, wound healing, and Annexin-V assay. We determined that β-Escin decreased glutamine uptake and reduced c-myc and GLS1 protein expressions and increased the expression of ASCT2. In addition, this inhibition of glutamine metabolism decreased cell proliferation, colony formation and migration, and induced apoptosis. In this study, it was suggested that β-Escin inhibits glutamine metabolism via c-myc in MDA-MB-231 cells, and it is thought that as a result of interrupting the energy supply in these cells via c-myc, it results in a decrease in the carcinogenic properties of the cells. Consequently, β-Escin may be promising as a therapeutic agent for glutamine-dependent cancers.

C-MYC and HIF1α promoter G-quadruplexes dependent metabolic regulation mechanism of berberine in colon cancer.

G-quadruplexes are molecular switches regulating gene transcription. c-MYC and hypoxia-inducible factor 1-alpha (HIF1α) play important roles in cell proliferation, apoptosis, and metabolic regulation in colon cancer. Whether berberine can regulate metabolism by interacting with c-MYC and HIF1α G-quadruplexes in colon cancer needs to be explored. The binding mode of berberine with c-MYC and HIF1α G-quadruplexes were explored by ultraviolet and visible absorption spectroscopy and fluorescence spectroscopy. Circular dichroism (CD) spectroscopy was performed to evaluate the effects of berberine on the stability of c-MYC and HIF1α G-quadruplexes. After different concentrations of berberine acting on HCT116 cells for 24 h, cell proliferation and apoptosis were detected by MTT assay and flow cytometry; quantitative real-time polymerase chain reaction and western blot were performed to detect mRNA and protein expression of c-MYC and HIF1α; transcriptome sequencing was used to analyze the metabolic pathways. For the effects of berberine on colon cancer mouse model with dose of 50 mg·kg-1 for 14 days, tumor growth were monitored, hematoxylin and eosin staining and immunofluorescence staining were performed to analyze histopathology and protein expression of c-MYC and HIF1α, central carbon metabolism was detected in tumor tissues. The binding ability of berberine with c-MYC G-quadruplex was different to that of berberine with HIF1α G-quadruplex. Both binding modes involved π-π stacking. The stoichiometric ratios were 1:1, 1:3, and 3:1 for berberine with c-MYC G-quadruplex and only 1:1 for berberine with HIF1α G-quadruplex. Temperature had a greater effect on the binding of berberine to c-MYC G-quadruplex. Berberine could improve the thermal stability of both c-MYC and HIF1α G-quadruplexes. Berberine inhibited the gene transcription and protein expression of c-MYC and HIF1α in colon cancer HCT116 cells. In vivo, berberine delayed tumor progression and inhibited the protein expression of c-MYC and HIF1α. Twelve differential metabolites such as decreased adenosine triphosphate were obtained, indicating that berberine could regulate the metabolic pathways of the tricarboxylic acid (TCA) cycle and glycolysis/gluconeogenesis, among others. Berberine may inhibit colon cancer by regulating the TCA cycle and glycolysis/gluconeogenesis based on the interaction with c-MYC and HIF1α G-quadruplexes.

Echinacoside Induces Mitochondria-Mediated Pyroptosis through Raf/MEK/ERK Signaling in Non-Small Cell Lung Cancer Cells.

Background Various natural compounds are effective in cancer prevention and treatment with fewer side effects than conventional radiotherapy and chemotherapy. Considering the uncertainty of the antitumor mechanism of Echinacoside (Ech) and the fact that no study on Ech against non-small cell lung cancer (NSCLC) has been explored previously, this study inquired into the anti-NSCLC effect of Ech and explored its potential mechanisms. Methods The IC50 to Ech of the NSCLC cells was calculated based on a series of cell viability assays. Different concentrations of Ech were used to treat the cells; the proliferation activity of the cells was evaluated using EdU staining. Mitochondrial membrane potential was detected by JC-1 staining. Levels of cytokines IL-1β and IL-18 were measured by ELISA. GSH and MDA levels were measured by microplate reader. Expression of cytochrome c, NLRP3, caspase-1, IL-1β, c-Myc, c-Fos, and Raf/MEK/ERK pathway proteins was evaluated by western blot. Meanwhile, we used xenograft, immunohistochemical staining, and H&E staining to evaluate the pharmacological effects of Ech in mice in vivo. Results ECH inhibited the proliferation of NSCLC cells. Ech increased the expression of pyroptosis-related proteins. Besides, Ech perturbed the mitochondrial membrane potential with the release of mitochondrial cytochrome c, accompanied by increased oxidative stress. Ech inhibited the phosphorylation levels of Raf/MEK/ERK signaling pathway and subsequently reduced c-myc and c-fos protein expression. In addition, Ech effectively restrained the growth of tumors in vivo. Conclusions Ech inhibited the Raf/MEK/ERK signaling. Impaired mitochondria activated inflammasome, which in turn led to the pyroptosis of NSCLC cells. These findings can provide some ideas on how to use pyroptosis to treat NSCLC.

Bone Marrow Mesenchymal Stem Cell (BMSC)-Exosomes Overexpressing miR-141 Inhibit the Malignant Biological Behavior of Glioma Cells via Wnt Signaling

Our study explores whether BMSC-exosomes overexpressing miR-141 can regulate Wnt signal to inhibit the malignant biological behavior of glioma cells. Thirty healthy mice were selected to construct a glioma mouse model and assigned randomly into the control group, miR-141 NC group, and miR-141 mimic group followed by analysis of cell proliferation, apoptosis, protein expression and mRNA expression by MTT method, flow cytometry, Western blot and RT-PCR methods. Compared with the other two groups, miR-141 mimic group showed reduced number of cell proliferation at 24 h and 48 h, decreased cell migration and invasion ability, and the increased cell apoptosis rate (P < 0.05). In miR-141 mimic group, the protein expression of miR-141 was the highest, while the protein expression of β-catenin, survivin and c-myc was the lowest (P < 0.05). In conclusion, BMSC-exosomes overexpressing miR-141 can inhibit the malignant biological behavior of GC cells possibly by inhibiting the activation of Wnt signaling pathway.

Role of the hydrogen sulfide-releasing donor ADT-OH in the regulation of mammal neural precursor cells.

Neural precursor cells (NPCs) generate new neurons to supplement neuronal loss as well as to repair damaged neural circuits. Therefore, NPCs have potential applications in a variety of neurological diseases, such as spinal cord injury, traumatic brain injury, and glaucoma. Specifically, improving NPCs proliferation and manipulating their differentiated cell types can be a beneficial therapy for a variety of these diseases. ADT-OH is a slow-releasing organic H2 S donor that produces a slow and continuous release of H2 S to maintain normal brain functions. In this study, we aimed to explore the effect of ADT-OH on NPCs. Our results demonstrated that ADT-OH promotes self-renewal and antiapoptosis ability of cultured NPCs. Additionally, it facilitates more NPCs to differentiate into neurons and oligodendrocytes, while inhibiting their differentiation into astrocytes. Furthermore, it enhances axonal growth. Moreover, we discovered that the mRNA and protein expression of β-catenin, TCF7L2, c-Myc, Ngn1, and Ngn2, which are key genes that regulate NPCs self-renewal and differentiation, were increased in the presence of ADT-OH. Altogether, these results indicate that ADT-OH may be a promising drug to regulate the neurogenesis of NPCs, and needs to be studied in the future for clinical application potential.

Study on the chemotherapeutic effect and mechanism of cucurbitacin E on laryngeal cancer stem cells.

ObjectivesStudy on the chemotherapeutic effect and mechanism of cucurbitacin E (CuE) on laryngeal cancer stem cells.MethodsWe used flow cytometry to sort out CD133+ laryngeal cancer stem cells; trypan blue rejection assay to detect the survival rate of laryngeal cancer stem cells; Cell counting kit‐8 (CCK‐8) assay to detect the effect of CuE on the proliferation ability of stem cells and the chemotherapeutic potentiation of doxorubicin; Transwell assay to observe the effect of CuE on the migration ability of stem cells; and Western Blot to detect the effect of CuE on the expression level of stem cell‐associated proteins. The tumor volume of nude mice was measured at the end of the experiment, and paraffin sections of nude mice tumor tissues were prepared and stained with Hematoxylin and eosin (H&E). The expression of c‐MYC in tumor tissues of nude mice was further detected by immunohistochemistry, and the effect of CuE on the expression level of related proteins in tumor tissues of nude mice was detected by Western Blot.ResultsCuE reduced the survival rate, proliferation ability, and migration ability of laryngeal cancer stem cells in vitro, and that CuE had a chemotherapeutic potentiating effect on doxorubicin. The possible mechanism of the chemotherapeutic effect of CuE was to reduce the expression of c‐MYC protein, and the possible mechanism of chemotherapy synergy was to reduce the expression of ABCG2 and P‐gp protein.ConclusionCuE has a chemotherapeutic effect on laryngeal cancer stem cells, as well as a chemotherapy synergy.

Targeting an MDM2/MYC Axis to Overcome Drug Resistance in Multiple Myeloma.

Simple SummaryWe show that high expression of MDM2 is associated with poor prognosis and enhanced drug resistance in human myeloma cell lines (HMCLs). Inhibition of MDM2 by RNAi or by the MDM2/XIAP dual inhibitor MX69 significantly increased the sensitivity of resistant HMCLs and primary MM samples to bortezomib and other anti-myeloma drugs, demonstrating that MDM2 can modulate drug response. We uncovered a novel oncogenic regulatory loop between MDM2 and c-Myc that mediated MM drug resistance. MDM2 inhibition resulted in a remarkable induction of apoptosis and suppression of relapsed MM cell growth. Mechanistically, MDM2 stabilized c-Myc mRNA to upregulate its expression, while c-Myc transcriptionally inducedMDM2. Targeting this regulatory loop with MX69 re-sensitized chemo-resistant MM cells to anti-myeloma agents, irrespective of TP53 tumor suppressor status, and prolonged survival in xenograft MM mouse model. These results establish a rationale for therapeutic targeting of the MDM2/c-Myc axis to improve the clinical outcome of patients with refractory/relapsed MM.Background: MDM2 is elevated in multiple myeloma (MM). Although traditionally, MDM2 negatively regulates p53, a growing body of research suggests that MDM2 plays several p53-independent roles in cancer pathogenesis as a regulator of oncogene mRNA stability and translation. Yet, the molecular mechanisms underlying MDM2 overexpression and its role in drug resistance in MM remain undefined. Methods: Both myeloma cell lines and primary MM samples were employed. Cell viability, cell cycle and apoptosis assays, siRNA transfection, quantitative real-time PCR, immunoblotting, co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), soft agar colony formation and migration assay, pulse-chase assay, UV cross-linking, gel-shift assay, RNA-protein binding assays, MEME-analysis for discovering c-Myc DNA binding motifs studies, reporter gene constructs procedure, gene transfection and reporter assay, MM xenograft mouse model studies, and statistical analysis were applied in this study. Results: We show that MDM2 is associated with poor prognosis. Importantly, its upregulation in primary MM samples and human myeloma cell lines (HMCLs) drives drug resistance. Inhibition of MDM2 by RNAi, or by the MDM2/XIAP dual inhibitor MX69, significantly enhanced the sensitivity of resistant HMCLs and primary MM samples to bortezomib and other anti-myeloma drugs, demonstrating that MDM2 can modulate drug response. MDM2 inhibition resulted in a remarkable suppression of relapsed MM cell growth, colony formation, migration and induction of apoptosis through p53-dependent and -independent pathways. Mechanistically, MDM2 was found to reciprocally regulate c-Myc in MM; MDM2 binds to AREs on c-Myc 3′UTR to increase c-Myc mRNA stability and translation, while MDM2 is a direct transcriptional target of c-Myc. MDM2 inhibition rendered c-Myc mRNA unstable, and reduced c-Myc protein expression in MM cells. Importantly, in vivo delivery of MX69 in combination with bortezomib led to significant regression of tumors and prolonged survival in an MM xenograft model. Conclusion: Our findings provide a rationale for the therapeutic targeting of MDM2/c-Myc axis to improve clinical outcome of patients with refractory/relapsed MM.

Gene Expression Profiling and Protein Analysis Reveal Suppression of the C-Myc Oncogene and Inhibition JAK/STAT and PI3K/AKT/mTOR Signaling by Thymoquinone in Acute Myeloid Leukemia Cells.

Overexpression of c-Myc plays an essential role in leukemogenesis and drug resistance, making c-Myc an attractive target for cancer therapy. However, targeting c-Myc directly is impossible, and c-Myc upstream regulator pathways could be targeted instead. This study investigated the effects of thymoquinone (TQ), a bioactive constituent in Nigella sativa, on the activation of upstream regulators of c-Myc: the JAK/STAT and PI3K/AKT/mTOR pathways in HL60 leukemia cells. Next-generation sequencing (NGS) was performed for gene expression profiling after TQ treatment. The expression of c-Myc and genes involved in JAK/STAT and PI3K/AKT/mTOR were validated by quantitative reverse transcription PCR (RT-qPCR). In addition, Jess assay analysis was performed to determine TQ’s effects on JAK/STAT and PI3K/AKT signaling and c-Myc protein expression. The results showed 114 significant differentially expressed genes after TQ treatment (p < 0.002). DAVID analysis revealed that most of these genes’ effect was on apoptosis and proliferation. There was downregulation of c-Myc, PI3K, AKT, mTOR, JAK2, STAT3, STAT5a, and STAT5b. Protein analysis showed that TQ also inhibited JAK/STAT and PI3K/AKT signaling, resulting in inhibition of c-Myc protein expression. In conclusion, the findings suggest that TQ potentially inhibits proliferation and induces apoptosis in HL60 leukemia cells by downregulation of c-Myc expression through inhibition of the JAK/STAT and PI3K/AKT signaling pathways.

Combination treatment of docetaxel with caffeic acid phenethyl ester suppresses the survival and the proliferation of docetaxel-resistant prostate cancer cells via induction of apoptosis and metabolism interference

BackgroundDocetaxel has been approved by USFDA as a first-line treatment for castration-resistant prostate cancer (CRPC) patients. Patients receiving androgen deprivation therapy along with docetaxel result in superior survival, lower serum prostate specific antigen (PSA) level, and better quality of life. However, a significant proportion of these patients ultimately develop resistance to docetaxel within months. Caffeic acid phenethyl ester (CAPE), one of the main bioactive components extracted from the propolis, has been reported to be effective for repressing the tumor growth, the migration and invasion of prostate cancer (PCa) cells, as well as the downstream signaling and stability of androgen receptor (AR). We hence determined if combination treatment of docetaxel with CAPE can suppress the proliferation and the survival of docetaxel-resistant PCa cells.MethodsWe established docetaxel-resistant PC/DX25 and DU/DX50 CRPC cell lines from PC-3 and DU-145 human PCa cells, respectively. Proliferation assay, MTT assay, flow cytometry with Annexin V staining, Comet Assay, and nude mice xenograft model were applied to determine the effects of combination treatment on cell proliferation and survival of the docetaxel-resistant PCa cells. Micro-Western Array (MWA) and qRT-PCR were used to investigate the molecular mechanism lying underneath.ResultsCombination treatment effectively suppressed the proliferation, survival and tumor growth of docetaxel-resistant PCa cells both in vitro and in nude mice. Comet assay and flow cytometry indicated that combination treatment induced apoptosis in docetaxel-resistant PCa cells. MWA and Western blotting assay revealed that combination treatment suppressed protein expression of Bcl-2, AKT2, c-Myc, apoptosis and caspase activation inhibitor (AVEN), pyruvate kinase M2 (PKM2) but increased protein expression of Bax, caspase 3, cytochrome c, glucose-6-phosphate dehydrogenase (G6PD) and acylglycerol kinase (AGK). Overexpression of Bcl-2 in the docetaxel-resistant PCa cells enhanced cell proliferation of docetaxel-resistant PCa cells under combination treatment. Analysis with qRT-PCR suggested that combination treatment decreased cholesterol biosynthesis genes DHCR24 (24-dehydrocholesterol reductase) and LSS (lanosterol synthase) but increased genes involved in glycolysis and TCA cycle.ConclusionsCombination treatment of docetaxel with CAPE effectively suppressed the proliferation and survival of docetaxel-resistant PCa cells via inhibition of Bcl-2 and c-Myc as well as induction of metabolism interference. Combination treatment can be beneficial for patients with docetaxel-resistant PCa.