Myb Oncogene Research Articles

68 (PB056): REM-422, a potent, selective, oral small moleculemRNA degrader of the MYB oncogene, induces regressions in mouse patient-derived xenograft models of adenoid cystic carcinoma

68 (PB056): REM-422, a potent, selective, oral small moleculemRNA degrader of the MYB oncogene, induces regressions in mouse patient-derived xenograft models of adenoid cystic carcinoma

Effect of RGT-61159 on inhibition of oncogene c-MYB synthesis and tumor growth inhibition in a broad range of ACC PDX models, at well tolerated doses in rodents and non-human primates.

6107 Background: RGT-61159 is a first in-class oral inhibitor of the oncogenic transcription factor c-MYB via selective alteration of its RNA splicing machinery. RGT-61159 selectively induces the inclusion of the cryptic “poison” exon into the c-MYB RNA transcripts, resulting in the robust elimination of c-MYB canonical mRNA transcript and consequently of its protein in cells. Overactivation of the c-MYB oncogene, primarily due to chromosome rearrangements, is a hallmark of adenoid cystic carcinoma. (ACC). ACC is a rare and aggressive type of cancer for which effective treatment does not exist. By inhibiting oncogenic MYB protein production, RGT-61159 has the potential to efficiently and selectively inhibit proliferation or induce cell death of cancer cells overexpressing MYB protein. Methods: RGT-61159 cell killing activity was measured against a broad panel of cancer cell lines in a CellTiter-Glo assay. MYB RNA and MYB protein levels were assessed after drug treatment by RT-qPCR and JESS assay respectively. Anti-tumor activity of RGT-61159 as single agent or in combination was evaluated in a panel of ACC PDX mouse models harboring different molecular alterations in c-MYB, including ACCX6, ACCX11, ACCx5M1 and ST105B2. Results: Here, we showed that RGT-61159 has potent cancer cell killing activity (EC50 – 100nM -200nM) against a large panel of cancer cell lines overexpressing c-MYB, while sparing normal cells. Robust c-MYB RNA and c-MYB protein depletion (>80%) was observed in treated cells confirming RGT-61159 on-target cell killing effect. In addition, RGT-61159 single agent showed a remarkable anti-tumor activity (up to 90% TGI) at tolerated doses in the four ACC PDX models evaluated: ACCX6, ACCx11, ACCx5M1 and STB105B2. Importantly, we confirmed that RGT-61159 anti-tumor activity correlated with c-MYB target modulation. RGT-61159 efficacious doses reduced c-MYB transcript and c-MYB protein levels (by >80%) in all ACC PDx models evaluated. In addition, RNAseq analysis of ACC tumor after treatment showed that RGT-61159 drug treatment reversed the published ACC overexpression gene signature. Notably, the optimal RGT-61159 regimen was well tolerated, as assessed by changes in animal body weight and clinical signs in rodents and non-human primate. Finally, we showed that the combination of RGT-M001, an analog of RGT-61159, with the NOTCH Inhibitor AL-101 resulted in significant tumor regression at well tolerated doses. Conclusions: Altogether, these data demonstrated that RGT-61159 efficiently and safely targets the undruggable oncogenic transcription factor c-MYB. Down-regulation of c-MYB by the RGT-61159 RNA-targeting small molecule is a very promising therapeutic strategy to treat ACC and other cancers driven by c-MYB dysregulation.

MYB alternative promoter activity is increased in adenoid cystic carcinoma metastases and is associated with a specific gene expression signature

ObjectiveAdenoid cystic carcinoma (ACC) is a head and neck cancer with a poor long-term prognosis that shows frequent local recurrences and distant metastases. The tumors are characterized by MYB oncogene activation and are notoriously unresponsive to systemic therapies. The biological underpinnings behind therapy resistance of disseminated ACC are largely unknown. Here, we have studied the molecular and clinical significance of MYB alternative promoter (TSS2) usage in ACC metastases. Materials and methodsMYB TSS2 activity was investigated in primary tumors and metastases from 26 ACC patients using RNA-sequencing and quantitative real-time PCR analysis. Differences in global gene expression between MYB TSS2 high and low cases were studied, and pathway analyses were performed. ResultsMYB TSS2 activity was significantly higher in ACC metastases than in primary tumors (median activity 15.1 vs 3.0, P = 0.0003). MYB TSS2 high ACC metastases showed a specific gene expression signature, including increased expression of multi-drug resistance genes and canonical MYB target genes, and suppression of the p53 and NOTCH pathways. ConclusionsCollectively, our findings indicate that elevated MYB TSS2 activity is associated with metastases, potential drug resistance, and augmented MYB-driven gene expression in ACC. Our study advocates the need for new therapies that specifically target MYB and drug resistance mechanisms in disseminated ACC.

Wnt/β-catenin-C-kit axis may play a role in adenoid cystic carcinoma prognostication

Adenoid cystic carcinoma (ACC) is one of the most common malignant salivary gland tumors. ACC is composed of myoepithelial and epithelial neoplastic cells which grow slowly and have a tendency for neural invasion. The long term prognosis is still relatively poor. Although several gene abnormalities, such as fusions involving MYB or MYBL1 oncogenes and the transcription factor gene NFIB, and overexpression of KIT have been reported in ACC, their precise functions in the pathogenesis of ACC remain unclear. We recently demonstrated that the elevated expression of Semaphorin 3A (SEMA3A), specifically expressed in myoepithelial neoplastic cells, might function as a novel oncogene-related molecule to enhance cell proliferation through activated AKT signaling in 9/10 (90%) ACC cases. In the current study, the patient with ACC whose tumor was negative for SEMA3A in the previous study, revisited our hospital with late metastasis of ACC to the cervical lymph node eight years after surgical resection of the primary tumor. We characterized this recurrent ACC, and compared it with the primary ACC using immunohistochemical methods. In the recurrent ACC, the duct lining epithelial cells, not myoepithelial neoplastic cells, showed an elevated Ki-67 index and increased cell membrane expression of C-kit, along with the expression of phosphorylated ERK. Late metastasis ACC specimens were not positive for β-catenin and lymphocyte enhancer binding factor 1 (LEF1), which were detected in the nuclei of perineural infiltrating cells in primary ACC cells. In addition, experiments with the GSK-3 inhibitor revealed that β-catenin pathway suppressed not only KIT expression but also proliferation of ACC cells. Moreover, stem cell factor (SCF; also known as KIT ligand, KITL) induced ERK activation in ACC cells. These results suggest that inactivation of Wnt/β-catenin signaling may promote C-kit-ERK signaling and cell proliferation of in metastatic ACC.

REM-422, a Potent, Selective, Oral Small Molecule mRNA Degrader of the MYB Oncogene, Demonstrates Anti-Tumor Activity in Mouse Xenograft Models of AML

The c-MYB (MYB) oncogenic transcription factor is a key regulator of hematopoietic cell differentiation and proliferation. Recurrent genetic lesions or dysregulation of MYB have been identified in a variety of cancers, including adenoid cystic carcinoma (ACC), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), pediatric low-grade glioma (pLGG), and others. AML is characterized by multiple oncogenic abnormalities and high MYB mRNA expression. Evidence from in vitro and in vivo animal studies suggests that MYB may be a key downstream effector of these oncogenic abnormalities through the direct regulation of genes such as BCL2, MYC and FLT3. Functional genomics studies reveal MYB dependencies in human leukemia cell lines and mouse models of leukemia. Together, these data suggest that MYB is an attractive therapeutic target for AML and other hematological malignancies. Therapeutic agents that directly target MYB have yet to be developed, presumably due to the challenges of targeting this intrinsically disordered transcription factor, which lacks tractable ligand binding pockets. Here, we describe the mechanism of action and biological activity of REM-422, a potent, selective, oral small molecule messenger RNA (mRNA) degrader of MYB. REM-422 was identified through screening and medicinal chemistry efforts directed towards identifying small molecules that induce the inclusion of a normally unused ‘poison exon’ (PE), which contains a premature termination codon, into the MYB pre-mRNA transcript. Poison exon inclusion activates the nonsense-mediated decay pathway and promotes the degradation of MYB mRNA. We demonstrate in a cell-free assay that REM-422 acts by selectively facilitating the binding of the U1 snRNP spliceosome complex, which governs exon usage by recognizing 5‘ splice sites, to oligonucleotides containing the MYB PE 5‘ splice site sequence. REM-422 treatment of human leukemia cell lines promotes poison exon inclusion, leading to degradation of MYB mRNA and subsequent reduction in MYB protein levels (Figure 1A). In a panel of AML cell lines, REM-422 demonstrated preferential anti-proliferative activity in cell lines with high MYB expression, and in those shown to be MYB-dependent through functional genomics studies. To understand the pharmacokinetic (PK) and pharmacodynamic (PD) effects of MYB in vivo, Kasumi-1 AML cells were implanted subcutaneously in CB17 scid immunocompromised mice and REM-422 was dosed by oral gavage after the formation of established tumors. QD PO administration of REM-422 demonstrated dose-dependent effects in tumors on MYB mRNA levels, with induction of the MYB poison exon leading to reductions of MYB functional mRNA. In a 21-day study, REM-422 demonstrated significant anti-tumor activity, including tumor regressions at well tolerated dose levels (Figure 1 left). To further investigate the activity of REM-422, AML patient cells were injected into NOG immunocompromised mice and allowed to engraft. After successful engraftment, mice were dosed by oral gavage once daily with REM-422 at 10 mg/kg or vehicle for 24 days. Flow cytometry analysis of bone marrow and peripheral blood samples demonstrated that REM-422 led to the eradication of human leukemia cells from both compartments (Figure 1 right). Together, these data demonstrate that REM-422 treatment leads to degradation of MYB mRNA in preclinical models of AML and supports the therapeutic potential for REM-422 in AML patients.

The Oncoprotein SKI As a Driver in KMT2A-MLLT3/NRasG12D-Positive Acute Myeloid Leukemia

KMT2A-rearranged acute myeloid leukemias (AML) have an intermediate to adverse prognosis. It was shown in a mouse model that the oncogene Myb contributes to AML maintenance in the KMT2A-MLLT3/NRasG12D genetic background (Zuber et al., 2011). Interestingly, the Myb-regulated genetic network mostly overlaps with KMT2A-MLLT3 target genes, especially with leukemic stem cell-associated gene sets. However, canonical KMT2A target genes are not included. Hence, MYB seems not to act as a co-factor for KMT2A complexes. SKI is a nuclear co-regulator and an oncoprotein, which is overexpressed in AML and several solid tumors (Bonnon & Atanasoski, 2012). Overexpression (OE) of SKI is prognostic in AML and correlates with shorter overall survival. Our group showed that SKI OE in AML is partially regulated by MYB (Frech et al., 2018). As a MYB target gene, we wanted to know if SKI can mediate MYB oncogenic activity in KMT2A-MLLT3/NRasG12D-positive AML. We adopted the Zuber mouse model and performed an shRNA-mediated Ski knockdown (KD) in murine KMT2A-MLLT3/NRasG12D-positive AML cells (RN2). Mice were transplanted with RN2 cells carrying two different anti-Ski shRNAs or an anti-Renilla shRNA as control. The expansion of RN2 cells in mice was monitored by luciferase-based bioimaging. Upon disease onset, shRNA expression was induced by doxycycline treatment. The course of the disease was further monitored by bioimaging. Bone marrow cells were isolated and analyzed via flow cytometry. The expression of KMT2A target genes was analyzed by RT-qPCR in doxycycline-induced RN2 cells. Ski KD induced a decreased viability of the RN2 cells in vitro. Moreover, SKI KD led to the eradication of RN2 cells in the mice in vivo and was associated with longer overall survival (log-rank p<0.0005). In line with the SKI OE mouse model of Singbrant et al. (2014), Ski KD led vice versa in our model to a decrease of myeloid cells and an increase of B cells. In contrast to Myb KD in the RN2 cells, Ski KD led to a lower expression of canonical KMT2A target genes, implicating that Ski may be part of KMT2A complexes in AML. In summary, SKI seems to be crucial for KMT2A-MLLT3/NRasG12D-driven AML and may be a potential target for new therapeutic strategies.

Increased MYB alternative promoter usage is associated with relapse in acute lymphoblastic leukemia.

Therapy-resistant disease is a major cause of death in patients with acute lymphoblastic leukemia (ALL). Activation of the MYB oncogene is associated with ALL and leads to uncontrolled neoplastic cell proliferation and blocked differentiation. Here, we used RNA-seq to study the clinical significance of MYB expression and MYB alternative promoter (TSS2) usage in 133 pediatric ALLs. RNA-seq revealed that all cases analyzed overexpressed MYB and demonstrated MYB TSS2 activity. qPCR analyses confirmed the expression of the alternative MYB promoter also in seven ALL cell lines. Notably, high MYB TSS2 activity was significantly associated with relapse (p = 0.007). Moreover, cases with high MYB TSS2 usage showed evidence of therapy-resistant disease with increased expression of ABC multidrug resistance transporter genes (e.g., ABCA2, ABCB5, and ABCC10) and enzymes catalyzing drug degradation (e.g., CYP1A2, CYP2C9, and CYP3A5). Elevated MYB TSS2 activity was further associated with augmented KRAS signaling (p < 0.05) and decreased methylation of the conventional MYB promoter (p < 0.01). Taken together, our results suggest that MYB alternative promoter usage is a novel potential prognostic biomarker for relapse and therapy resistance in pediatric ALL.

Synthetic oleanane triterpenoids suppress MYB oncogene activity and sensitize T-cell acute lymphoblastic leukemia cells to chemotherapy.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with poor prognosis. The MYB oncogene encodes a master transcription factor that is activated in the majority of human T-ALLs. In the present study, we have performed a large-scale screening with small-molecule drugs to find clinically useful inhibitors of MYB gene expression in T-ALL. We identified several pharmacological agents that potentially could be used to treat MYB-driven malignancies. In particular, treatment with the synthetic oleanane triterpenoids (OTs) bardoxolone methyl and omaveloxolone decreased MYB gene activity and expression of MYB downstream target genes in T-ALL cells with constitutive MYB gene activation. Notably, treatment with bardoxolone methyl and omaveloxolone led to a dose-dependent reduction in cell viability and induction of apoptosis at low nanomolar concentrations. In contrast, normal bone marrow-derived cells were unaffected at these concentrations. Bardoxolone methyl and omaveloxolone treatment downregulated the expression of DNA repair genes and sensitized T-ALL cells to doxorubicin, a drug that is part of the standard therapy of T-ALL. OT treatment may thus potentiate DNA-damaging chemotherapy through attenuation of DNA repair. Taken together, our results indicate that synthetic OTs may be useful in the treatment of T-ALL and potentially also in other MYB-driven malignancies.

Abstract 1409: Synergistic antitumorigenic activity of Berberine and oligomeric proanthocyanidins through regulation of PI3-AKt signaling pathway in colorectal cancer

Abstract Background: The development of chemo-resistance against conventional chemotherapeutic drugs presents a major clinical challenge in the management of patients with colorectal cancer (CRC). Accumulating body of data in recent years have shown that active principles within various naturally-occurring dietary botanicals not only offer time-tested safety and anti-cancer efficacy, but a combination of certain compounds can overcome the elusive chemotherapeutic resistance in cancer patients. In this regard, data in recent years have shown that berberine (BBR) and oligomeric proanthocyanidins (OPCs) from the grape-seed extract have significant anti-tumorigenic properties in CRC, and a more recent study reported existence of synergism between these two natural medicines in patients with type 2 diabetes. Accordingly, herein we hypothesized that BBR and OPCs might regulate synergistically multiple oncogenic pathways to exert a superior anti-cancer activity in CRC. Methods: We performed a series of experiment in various cell lines, followed by their interrogation in patient-derived organoids (PDOs) to evaluate the synergistic anti-tumorigenic effects of BBR and OPCs in CRC cells. In addition, using genome-wide RNA sequencing analyses, we identified specific functional determinants and key targeted genes and pathways regulated by the combined therapeutic approach with these compounds. Results: To confirm our hypothesis, we first evaluated the potential of OPCs to increase the cellular uptake of BBR in CRC cells, by measuring the fluorescent signal of BBR in multiple cell lines treated individually or their combination. The synergistic anti-tumorigenic effects of BBR and OPCs were observed in various functional assays including reduced cell viability, colony formation potential, wound healing, and invasion assays. Furthermore, the combined treatment with these compounds potentiated the cell apoptosis in an Annexin V binding assay. It was intriguing to note that we were able to successfully validate all cell culture findings in the PDO models from multiple CRC patients. Whole genome transcriptomic profiling identified oncogene MYB, including in PI3-AKT signaling pathway as one of the key pathways that was critically involved in mediating the anti-tumorigenic properties of this combined therapy. Conclusions: We for the first time demonstrate that a combined treatment with BBR and OPCs synergistically promotes the anti-tumorigenic properties in CRC. This synergism was potentially due to enhanced apoptosis and the regulation of oncogenic MYB in the PI3-Akt signaling pathway; hence, offering an attractive potential for the use of these two compounds as adjuvant treatment options in patients with CRC. Citation Format: Keisuke Okuno, Rachana Garg, Masanori Tokunaga, Yusuke Kinugasa, Ajay Goel. Synergistic antitumorigenic activity of Berberine and oligomeric proanthocyanidins through regulation of PI3-AKt signaling pathway in colorectal cancer [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 1409.

Berberine and Oligomeric Proanthocyanidins Exhibit Synergistic Efficacy Through Regulation of PI3K-Akt Signaling Pathway in Colorectal Cancer.

BackgroundNaturally occurring dietary botanicals offer time-tested safety and anti-cancer efficacy, and a combination of certain compounds has shown to overcome the elusive chemotherapeutic resistance, which is of great significance for improving the mortality of patients with colorectal cancer (CRC). Accordingly, herein, we hypothesized that berberine (BBR) and oligomeric proanthocyanidins (OPCs) might regulate synergistically multiple oncogenic pathways to exert a superior anti-cancer activity in CRC.MethodsWe performed a series of cell culture studies, followed by their interrogation in patient-derived organoids to evaluate the synergistic effect of BBR and OPCs against CRC. In addition, by performing whole genome transcriptomic profiling we identified the key targeted genes and pathways regulated by the combined treatment.ResultsWe first demonstrated that OPCs facilitated enhanced cellular uptake of BBR in CRC cells by measuring the fluorescent signal of BBR in cells treated individually or their combination. The synergism between BBR and OPCs were investigated in terms of their anti-tumorigenic effect on cell viability, clonogenicity, migration, and invasion. Furthermore, the combination treatment potentiated the cellular apoptosis in an Annexin V binding assay. Transcriptomic profiling identified oncogene MYB in PI3K-AKT signaling pathway might be critically involved in the anti-tumorigenic properties of the combined treatment. Finally, we successfully validated these findings in patient-derived CRC tumor organoids.ConclusionsCollectively, we for the first time demonstrate that a combined treatment of BBR and OPCs synergistically promote the anti-tumorigenic properties in CRC possibly through the regulation of cellular apoptosis and oncogene MYB in the PI3K-Akt signaling pathway.

Proteasome inhibitors suppress MYB oncogenic activity in a p300-dependent manner.

Studies of the role of MYB in human malignancies have highlighted MYB as a potential drug target for acute myeloid leukemia (AML) and adenoid cystic carcinoma (ACC). Although transcription factors are often considered un-druggable, recent work has demonstrated successful targeting of MYB by low molecular weight compounds. This has fueled the notion that inhibition of MYB has potential as a therapeutic approach against MYB-driven malignancies. Here, we have used a MYB reporter cell line to screen a library of FDA-approved drugs for novel MYB inhibitors. We demonstrate that proteasome inhibitors have significant MYB-inhibitory activity, prompting us to characterize the proteasome inhibitor oprozomib in more detail. Oprozomib was shown to interfere with the ability of the co-activator p300 to stimulate MYB activity and to exert anti-proliferative effects on human AML and ACC cells. Overall, our work demonstrated suppression of oncogenic MYB activity as a novel result of proteasome inhibition.

Convergent organization of aberrant MYB complex controls oncogenic gene expression in acute myeloid leukemia.

Dysregulated gene expression contributes to most prevalent features in human cancers. Here, we show that most subtypes of acute myeloid leukemia (AML) depend on the aberrant assembly of MYB transcriptional co-activator complex. By rapid and selective peptidomimetic interference with the binding of CBP/P300 to MYB, but not CREB or MLL1, we find that the leukemic functions of MYB are mediated by CBP/P300 co-activation of a distinct set of transcription factor complexes. These MYB complexes assemble aberrantly with LYL1, E2A, C/EBP family members, LMO2, and SATB1. They are organized convergently in genetically diverse subtypes of AML and are at least in part associated with inappropriate transcription factor co-expression. Peptidomimetic remodeling of oncogenic MYB complexes is accompanied by specific proteolysis and dynamic redistribution of CBP/P300 with alternative transcription factors such as RUNX1 to induce myeloid differentiation and apoptosis. Thus, aberrant assembly and sequestration of MYB:CBP/P300 complexes provide a unifying mechanism of oncogenic gene expression in AML. This work establishes a compelling strategy for their pharmacologic reprogramming and therapeutic targeting for diverse leukemias and possibly other human cancers caused by dysregulated gene control.

ATR is a MYB regulated gene and potential therapeutic target in adenoid cystic carcinoma

Adenoid cystic carcinoma (ACC) is a rare cancer that preferentially occurs in the head and neck, breast, as well as in other sites. It is an aggressive cancer with high rates of recurrence and distant metastasis. Patients with advanced disease are generally incurable due to the lack of effective systemic therapies. Activation of the master transcriptional regulator MYB is the genomic hallmark of ACC. MYB activation occurs through chromosomal translocation, copy number gain or enhancer hijacking, and is the key driving event in the pathogenesis of ACC. However, the functional consequences of alternative mechanisms of MYB activation are still uncertain. Here, we show that overexpression of MYB or MYB-NFIB fusions leads to transformation of human glandular epithelial cells in vitro and results in analogous cellular and molecular consequences. MYB and MYB-NFIB expression led to increased cell proliferation and upregulation of genes involved in cell cycle control, DNA replication, and DNA repair. Notably, we identified the DNA-damage sensor kinase ATR, as a MYB downstream therapeutic target that is overexpressed in primary ACCs and ACC patient-derived xenografts (PDXs). Treatment with the clinical ATR kinase inhibitor VX-970 induced apoptosis in MYB-positive ACC cells and growth inhibition in ACC PDXs. To our knowledge, ATR is the first example of an actionable target downstream of MYB that could be further exploited for therapeutic opportunities in ACC patients. Our findings may also have implications for other types of neoplasms with activation of the MYB oncogene.

Genetic Polymorphisms of HBS1L-MYB (rs4895441 and rs9376090) in Egyptian Patients with Hemoglobinopathy

Objective: Study the HBS1L-MYB (rs4895441 and rs9376090) genetic polymorphisms in Egyptian patients with β-thalassemia major and sickle cell disease and its relation to Hb F and severity of the disease. Background: Hb F is a predominant modulator for the severity of β-thalassemia major & sickle cell disease. Genetic polymorphism in the intergenic region (HBS1L-MYB) between GTP-binding elongation factor HBS1L and myeloblastosis oncogene MYB on chromosome 6q is associated with high fetal hemoglobin levels. Subjects and Methods: 150 subjects were included in this study. For all studied groups: Complete blood picture and serum ferritin were evaluated. For patients, hemoglobin variants were separated by High-performance liquid chromatography. Genotyping of HBS1L-MYB (rs4895441 & rs9376090) was evaluated by real-time polymerase chain reaction technique using TaqMan probe. Results: AG, CT genotypes, and G, C alleles of HBS1L-MYB (rs4895441 & rs9376090) were significantly high in sickle cell patients [OR (3.400); 95% C.I (1.482 - 7.799)], (p = 0.003) & [OR (4.522); 95% C.I (1.854 - 11.029)], (p = 0.001) respectively. Also, a significant association was detected between polymorphisms and disease severity. However, in β-thalassemia major, no significant association was detected. Conclusion: In sickle cell disease patients, Genetic polymorphisms in HBS1L-MYB (rs9376090 & rs4895441) affect the level of Hb F which could improve the prognosis of these patients.

N-Terminal Truncated Myb with New Transcriptional Activity Produced Through Use of an Alternative MYB Promoter in Salivary Gland Adenoid Cystic Carcinoma.

Adenoid cystic carcinoma (ACC) is an aggressive salivary gland tumor that frequently displays perineural invasion and is often associated with translocations or overexpression of the MYB oncogene. Detailed analyses of MYB transcripts from ACC patient samples revealed that ACC tumors utilize an alternative MYB promoter, which is rarely used in normal cells or other tumor types. The alternative promoter transcripts produce N-terminally truncated Myb proteins lacking a highly conserved and phosphorylated domain, which includes the pS11 epitope that is frequently used to detect Myb proteins. In RNA-seq assays, Myb isoforms lacking the N-terminal domain displayed unique transcriptional activities, regulating many genes differently than full-length Myb. Thus, a regulatory pathway unique to ACC activates the alternative MYB promoter, leading to the production of a truncated Myb protein with altered transcriptional activities. This could provide new therapeutic opportunities for ACC patients.

Clinical, genetic and experimental studies of the Brooke–Spiegler (CYLD) skin tumor syndrome

Brooke–Spiegler syndrome (BSS; a.k.a. tuban tumor syndrome) is an autosomal dominant inherited skin disorder caused by germline mutations in the CYLD tumor suppressor gene. BSS is characterized by multiple skin adnexal tumors, mainly cylindromas and spiradenomas on the head and neck. The tumors are often severely disfiguring and require repeated surgical interventions. Here, we describe a four-generation BSS-family with a novel germline c.1613_1614delGC CYLD mutation that introduces a premature STOP codon predicted to result in a truncated, inactivated CYLD protein. In addition, we present a pilot study describing establishment of the first patient-derived xenografts (PDXs) from cutaneous CYLD-defective cylindromas. Fresh tumor tissues from cylindromas were transplanted into immunocompromised mice to generate PDXs. One xenograft showed progressive tumor growth after 3 months whereas the others remained unchanged in size during the 6 months study period. Histopathological and immunohistochemical analyses of the PDXs revealed that they recapitulate the histological and molecular features of their respective primary tumors, including expression of NTRK3 and the oncogenic driver MYB. In summary, we present the first preclinical BSS-model that morphologically and genetically recapitulates human CYLD-defective cylindromas. This model will be useful for preclinical therapeutic drug testing and for further studies of the molecular pathogenesis of inherited cylindromas.

Abstract 2758: Transcriptomics analysis of SNX-2112 on immune and mitochodrial genes

Abstract Introduction: SNX-2112 is a potent anti-tumor compound with effects on epigenetics (hypomethylation) and onco-metabolism. SNX-5422 is the orally active prodrug of SNX-2112, a highly selective inhibitor of heat-shock protein 90 (Hsp90) that has shown anti-tumor activity in clinical trials. Hsp90 is commonly elevated in tumor tissues and plays an important role in immune responses. Previous investigations have shown that SNX-2112 is highly active in TP53 null tumors and has good activity in combination with checkpoint inhibitors in syngeneic mice studies. Preclinical studies have suggested that Hsp90 inhibitors enhance antigen-specific T-cell recognition of cancer cells, increase the expression of both differentiation and MHC Class I antigens, and do not interfere with T-cell function. Methods: In two in vitro studies, the effects of SNX-2112 on immune (A375 melanoma cell line and the TP53 mutant chronic myelocytic leukemia K562 cell line) and mitochondrial genes (K652) using transcriptomics were investigated. A375 and K562 cells were cultured in media (GIBCO or Sigma, USA) supplemented with 10% fetal bovine serum at 37°C, 5% CO2, and 95% humidity. Data from transcriptomics were obtained, and affected genes analyzed. Results: In A375 cells, transcriptomics analysis revealed that SNX-2112 downregulated multiple overexpressed immune-related genes (including NFKB1, NFKB2, BRAF, HLA-DMA, HLA-DMB, HLA-DOB, HLA-DQB1, and HLA-DRB6) and upregulated multiple suppressed immune-related genes (including IFIT1, IFIT2, IFIT3, MLANA, DDX58, PMEL (gp100), DCT (TRP-2), MR1, MICA, and MICB). Transcriptomics analysis of SNX-2112 on K562 immune genes revealed that IFIT1-3, IFI6, IFI44, ISG15, DDX-58, and HLA-DRA were all upregulated. Genes downregulated by SNX-2112 included NFKB and IKBKB (Ikk), HLA-DOB, HLA-DPA1, HLA-DPB1, HLA-DQB1, PSMB8, and TAP1. In both the A375 and K562 cells lines, SNX-2112 reduced the expression of MYB oncogene, (reported to control regulatory T-cell proliferation) and caused increases in RB1CC1 (fip200) and STAT1, crucial survival factors for naïve T-cells. Additionally, tryptophan metabolism genes IDO1 and TDO2 were downregulated by SNX-2112. K562 mitochondrial genes downregulated by SNX-2112 included the following: •TP53 and p53 family members-controlled markers: HK2, GLS2, TIGAR •Glutamine addiction/Warburg effect: SLC1A5, GLUD1 •Folate pathway: SLC19A1, TYMS, SHMT1, AS3MT, DHFR2 •OXPHOS: CISD1, CHCHD4 Conclusion: SNX-2112 increased multiple suppressed immune-related genes and downregulated multiple overexpressed immune-related genes in A375 and K562 cell lines. Anti-tumor activity of SNX-2112 appears to be, in part, the result of interference with onco-metabolic pathways. Inhibition of IDO1 and TDO2 suggests possibly enhanced anti-tumor activity in combination with checkpoint inhibitors. Citation Format: Everardus Orlemans, Steven E. Hall. Transcriptomics analysis of SNX-2112 on immune and mitochodrial genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2758.

RUNX1 is required for oncogenic Myb and Myc enhancer activity in T-cell acute lymphoblastic leukemia

AbstractThe gene encoding the RUNX1 transcription factor is mutated in a subset of T-cell acute lymphoblastic leukemia (T-ALL) patients, and RUNX1 mutations are associated with a poor prognosis. These mutations cluster in the DNA-binding Runt domain and are thought to represent loss-of-function mutations, indicating that RUNX1 suppresses T-cell transformation. RUNX1 has been proposed to have tumor suppressor roles in T-cell leukemia homeobox 1/3–transformed human T-ALL cell lines and NOTCH1 T-ALL mouse models. Yet, retroviral insertional mutagenesis screens identify RUNX genes as collaborating oncogenes in MYC-driven leukemia mouse models. To elucidate RUNX1 function(s) in leukemogenesis, we generated Tal1/Lmo2/Rosa26-CreERT2Runx1f/f mice and examined leukemia progression in the presence of vehicle or tamoxifen. We found that Runx1 deletion inhibits mouse leukemic growth in vivo and that RUNX silencing in human T-ALL cells triggers apoptosis. We demonstrate that a small molecule inhibitor, designed to interfere with CBFβ binding to RUNX proteins, impairs the growth of human T-ALL cell lines and primary patient samples. We demonstrate that a RUNX1 deficiency alters the expression of a crucial subset of TAL1- and NOTCH1-regulated genes, including the MYB and MYC oncogenes, respectively. These studies provide genetic and pharmacologic evidence that RUNX1 has oncogenic roles and reveal RUNX1 as a novel therapeutic target in T-ALL.

Abstract 1990: Unique in vitro transcriptional activities of oncogenic Myb fusions identified in salivary gland adenoid cystic carcinoma

Abstract Chromosomal translocations involving the MYB and NFIB (t(6;9)) genes are the sole recurrent cytogenetic aberration in Adenoid Cystic Carcinoma (ACC), a rare cancer that frequently manifests as salivary gland tumors. Since ACC has poor prognosis and treatment is accompanied by high morbidity, there is a need for additional investigation of this cancer with the goal of improving patient outcomes. Recurrent translocations create fusions involving the N-terminus of MYB, located on chromosome 6, and the C-terminus of NFIB, located on chromosome 9. Our lab also recently identified translocations involving the related MYBL1 gene fusing it to NFIB t(8;9) and RAD51B t(8;14) genes. Further, recent work in our lab demonstrated tumors with MYB or MYBL1 translocations have similar gene expression (1). The presence of recurrent translocations involving both the MYB proto-oncogene and the related MYBL1 gene suggests that the resulting novel fusion proteins are likely oncogenic drivers in these tumors. In order to directly address the transcriptional activities of the resulting fusion proteins MYB and MYBL1 fusion genes, identified in primary ACC tumors, were cloned and expressed in vitro. The fusion proteins transcriptional activities were examined using reporter gene assays and trancriptomics. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed to interrogate promoters bound by these proteins. Reporter genes specific for normal vs. oncogenic MYB variants have been constructed. These data demonstrate that the proteins predicted to be produced by MYB fusion genes after chromosomal translocation are transcriptionally active. Further, Myb fusion proteins bind and activate gene sets distinct from their wild type c-Myb and A-Myb counterparts. These data indicate Myb fusion proteins have unique transcriptional activities, perhaps underlying their ability to drive tumorigensis in ACC. 1. Brayer et al. Recurrent Fusions in MYB and MYBL1 Define a Common, Transcription Factor-Driven Oncogenic Pathway in Salivary Gland Adenoid Cystic Carcinoma. Cancer Discovery. In press. Citation Format: Candace A. Frerich, Scott A. Ness. Unique in vitro transcriptional activities of oncogenic Myb fusions identified in salivary gland adenoid cystic carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1990.

An oncogenic MYB feedback loop drives alternate cell fates in adenoid cystic carcinoma.

Translocation events are frequent in cancer and may create chimeric fusions or ‘regulatory rearrangements’ that drive oncogene overexpression. Here we identify super-enhancer translocations that drive overexpression of the oncogenic transcription factor MYB as a recurrent theme in adenoid cystic carcinoma (ACC). Whole-genome sequencing data and chromatin maps reveal distinct chromosomal rearrangements that juxtapose super-enhancers to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers interact with the MYB promoter. Remarkably, MYB protein binds to the translocated enhancers, creating a positive feedback loop that sustains its expression. MYB also binds enhancers that drive different regulatory programs in alternate cell lineages in ACC, cooperating with TP63 in myoepithelial cells and a Notch program in luminal epithelial cells. Bromodomain inhibitors slow tumor growth in ACC primagraft models in vivo. Thus, our study identifies super-enhancer translocations that drive MYB expression and provides insight into downstream MYB functions in the alternate ACC lineages.