Pei-Yu Kuo

High-resolution chromatin immunoprecipitation (ChIP) sequencing reveals novel binding targets and prognostic role for SOX11 in mantle cell lymphoma

Sex determining region Y-box 11 (SOX11) expression is specific for mantle cell lymphoma (MCL) as compared with other non-Hodgkin’s lymphomas. However, the function and direct-binding targets of SOX11 in MCL are largely unknown. We used high-resolution chromatin immunoprecipitation sequencing to identify the direct target genes of SOX11 in a genome-wide, unbiased manner and elucidate its functional significance. Pathway analysis identified WNT, PKA and TGF-beta signaling pathways as significantly enriched by SOX11-target genes. Quantitative chromatin immunoprecipitation sequencing and promoter reporter assays confirmed that SOX11 directly binds to individual genes and modulates their transcription activities in these pathways in MCL. Functional studies using RNA interference demonstrate that SOX11 directly regulates WNT in MCL. We analyzed SOX11 expression in three independent well-annotated tissue microarrays from the University of Wisconsin (UW), Karolinska Institute and British Columbia Cancer Agency. Our findings suggest that high SOX11 expression is associated with improved survival in a subset of MCL patients, particularly those treated with intensive chemotherapy. Transcriptional regulation of WNT and other biological pathways affected by SOX11-target genes may help explain the impact of SOX11 expression on patient outcomes.

Integrative Genomic Analysis of Temozolomide Resistance in Diffuse Large B-Cell Lymphoma

Purpose: Despite advances, there is an urgent need for effective therapeutics for relapsed diffuse large B-cell lymphoma, particularly in elderly patients and primary central nervous system (CNS) lymphoma. Temozolomide (TMZ), an oral DNA-alkylating agent routinely used in the therapy of glioblastoma multiforme, is active in patients with primary CNS lymphoma but the response rates are low. The mechanisms contributing to TMZ resistance are unknown. Experimental Design: We undertook an unbiased and genome-wide approach to understand the genomic methylation and gene expression profiling differences associated with TMZ resistance in diffuse large B-cell lymphoma cell lines and identify mechanisms to overcome TMZ resistance. Results: TMZ was cytotoxic in a subset of diffuse large B-cell lymphoma cell lines, independent of MGMT promoter methylation or protein expression. Using Connectivity Map (CMAP), we identified several compounds capable of reversing the gene expression signature associated with TMZ resistance. The demethylating agent decitabine (DAC) is identified by CMAP as capable of reprogramming gene expression to overcome TMZ resistance. Treatment with DAC led to increased expression of SMAD1, a transcription factor involved in TGF-β/bone morphogenetic protein (BMP) signaling, previously shown to be epigenetically silenced in resistant diffuse large B-cell lymphoma. In vitro and in vivo treatment with a combination of DAC and TMZ had greater antilymphoma activity than either drug alone, with complete responses in TMZ-resistant diffuse large B-cell lymphoma murine xenograft models. Conclusions: Integrative genome-wide methylation and gene expression analysis identified novel genes associated with TMZ resistance and demonstrate potent synergy between DAC and TMZ. The evidence from cell line and murine experiments supports prospective investigation of TMZ in combination with demethylating agents in diffuse large B-cell lymphoma. Clin Cancer Res; 20(2); 382–92. ©2013 AACR.

Harnessing Noxa demethylation to overcome Bortezomib resistance in mantle cell lymphoma

Bortezomib (BZM) is the first proteasome inhibitor approved for relapsed Mantle Cell Lymphoma (MCL) with durable responses seen in 30%–50% of patients. Given that a large proportion of patients will not respond, BZM resistance is a significant barrier to use this agent in MCL. We hypothesized that a subset of aberrantly methylated genes may be modulating BZM response in MCL patients. Genome-wide DNA methylation analysis using a NimbleGen array platform revealed a striking promoter hypomethylation in MCL patient samples following BZM treatment. Pathway analysis of differentially methylated genes identified molecular mechanisms of cancer as a top canonical pathway enriched among hypomethylated genes in BZM treated samples. Noxa, a pro-apoptotic Bcl-2 family member essential for the cytotoxicity of BZM, was significantly hypomethylated and induced following BZM treatment. Therapeutically, we could demethylate Noxa and induce anti-lymphoma activity using BZM and the DNA methytransferase inhibitor Decitabine (DAC) and their combination in vitro and in vivo in BZM resistant MCL cells. These findings suggest a role for dynamic Noxa methylation for the therapeutic benefit of BZM. Potent and synergistic cytotoxicity between BZM and DAC in vitro and in vivo supports a strategy for using epigenetic priming to overcome BZM resistance in relapsed MCL patients.

Dual Targeting of CDK4 and ARK5 Using a Novel Kinase Inhibitor ON123300 Exerts Potent Anticancer Activity against Multiple Myeloma

Multiple myeloma is a fatal plasma cell neoplasm accounting for over 10,000 deaths in the United States each year. Despite new therapies, multiple myeloma remains incurable, and patients ultimately develop drug resistance and succumb to the disease. The response to selective CDK4/6 inhibitors has been modest in multiple myeloma, potentially because of incomplete targeting of other critical myeloma oncogenic kinases. As a substantial number of multiple myeloma cell lines and primary samples were found to express AMPK-related protein kinase 5(ARK5), a member of the AMPK family associated with tumor growth and invasion, we examined whether dual inhibition of CDK4 and ARK5 kinases using ON123300 results in a better therapeutic outcome. Treatment of multiple myeloma cell lines and primary samples with ON123300 in vitro resulted in rapid induction of cell-cycle arrest followed by apoptosis. ON123300-mediated ARK5 inhibition or ARK5-specific siRNAs resulted in the inhibition of the mTOR/S6K pathway and upregulation of the AMPK kinase cascade. AMPK upregulation resulted in increased SIRT1 levels and destabilization of steady-state MYC protein. Furthermore, ON123300 was very effective in inhibiting tumor growth in mouse xenograft assays. In addition, multiple myeloma cells sensitive to ON123300 were found to have a unique genomic signature that can guide the clinical development of ON123300. Our study provides preclinical evidence that ON123300 is unique in simultaneously inhibiting key oncogenic pathways in multiple myeloma and supports further development of ARK5 inhibition as a therapeutic approach in multiple myeloma.

Abstract B062: Differences of transcriptional profiles between long-term tolerized and memory CD4 T cells

It is well known that immunological adjuvants improve antigen-specific immune responses to cancer vaccines. We previously reported a clinical trial in non-small cell lung cancer where half of the patients received a cycle of 4 vaccines every 3 weeks with recombinant MAGEA3 protein alone in the absence of adjuvant, while the other half were vaccinated with MAGEA3 protein administered in adjuvant AS02B containing saponin and TLR4-ligand Monophosphoryl Lipid A. Results showed that vaccination without adjuvant led to very limited or no humoral and T cell immune responses to MAGEA3, while vaccine with adjuvant elicited robust humoral and CD4 T cell responses. Two to three years later, patients received a second cycle of vaccination, but this time with AS02B adjuvant for everyone. Remarkably, patients initially vaccinated without adjuvant still failed to mount immune responses even after 4 vaccines with adjuvant, exhibiting a tolerized phenotype, while remaining patients had a typical recall memory response reaching a plateau of high antibody and CD4 T cell levels after a single injection. This data suggested that the initial environment during priming dictates subsequent long-term capacity of CD4 T cells to expand and produce cytokines. While long-term tolerized CD4 T cells were induced in patients primed without adjuvant, in contrast to long-term memory CD4 T cells in patients vaccinated with adjuvant throughout, markers associated with T cell responsiveness vs. tolerance remain unclear. Here, we assessed antigen-specific cells using RNASeq analyses to elucidate at the transcriptional level mechanisms associated with qualitative differences in the CD4 T cell repertoire We applied a sensitive method based on CD154 upregulation to detect and sort MAGEA3-specific CD4 T cells throughout the study, including even rare precursors present at baseline, from two patients representative of tolerized vs. memory profile respectively. Unexpectedly, we found that the number of CD154-expressing cells after MAGEA3 in vitro restimulation were highly increased in both patients after the first vaccine injection, but durably reduced thereafter in the tolerized patient, while still increasing in the responder patient. To characterize differences in transcriptional profiles, MAGEA3 specific CD4 T cells were subjected to RNASeq at baseline and after the 1st and 2nd immunization. Sorted MAGEA3 specific CD4 T cells were non-specifically expanded, and restimulated with MAGEA3, followed by RNA isolation and deep sequencing by RNASeq. A total of ~16,000 individual genes were found transcribed in all samples, and many were related to expected CD4 T cell characteristics. When expression levels of all genes were considered together in unsupervised principal component analysis, there was a remarkable difference in profiles between the two patients. While the responder had a consistent shift in gene transcript post-vaccine compared to baseline, the tolerized patient had a transient shift in genes expressed by MAGEA3-specific CD4 T cells after one vaccine, which was abolished and returned to a profile closer to baseline after the 2nd vaccine. We identified a set of 413 genes with differential expression profiles between tolerized and responder patients over early vaccination time points. These genes can be further classified according to whether they increase or decrease specifically in the tolerized or in the responder patient, when comparing baseline to 1st vaccine to 2nd vaccine, and by their constitutive vs. inducible nature following antigen stimulation. Immune-related genes were heavily predominant within this subset, and we found an array of cytokines and checkpoint molecules that we propose as candidates for a tolerance signature. These results could be useful for early prediction of vaccine efficacy and patient benefit in future immunotherapy trials. Citation Format: Naoko Imai, Takemasa Tsuji, Nasser K. Altorki, Pei-Yu Kuo, Deepak Perumal, Samir Parekh, Sacha Gnjatic. Differences of transcriptional profiles between long-term tolerized and memory CD4 T cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B062.

Abstract 1324: Dual targeting of CDK4 and ARK5 using a novel kinase inhibitor ON123300 is effective in vitro and in vivo in Multiple Myeloma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Multiple Myeloma (MM) is an incurable malignancy of plasma cells afflicting over 21,000 patients and accounting for over 10,000 deaths in the US each year. It is characterized by recurrent chromosomal translocations resulting in the dysregulation of cyclinD1, cyclinD3, c-Maf, MafB and MMSET. Despite cell cycle dysregulation being prominent in myeloma pathogenesis, clinical efficacy of CDK4/CDK6 inhibitors has been modest. We therefore developed ON123300, an orally available and potent small inhibitor of CDK4/CDK6 and ARK5. ARK5 is a member of the AMP-activated protein kinase (AMPK) catalytic subunit family and is associated with increased tumor cell invasiveness in MM [Suzuki et al., Mol. Cell. Bio 2004] and transcriptionally regulated by oncogenes such as MMSET, c-Maf and MafB. In this study, we evaluated the in vitro and in vivo efficacy of ON123300 in MM cell lines. A panel of nine MM cell lines was treated with varying doses of ON123300 and cell viability assessed using the CellTitre Blue assay after 48 hours. ON123300 decreased cell viability by approximately 30 to 70% (IC50:50-200nM) in 9/9 MM cell lines examined. Based on IC50 of 50 nM, we could stratify the cell lines into two groups: 6 cell lines (MM.1R, MM.1S, KMS11, U266, RPMI-8226 and ARP1) had IC50 of 50-150 nm and three cell lines (EJM, JJN3 and NCI-H929) were very sensitive (IC50 <50nM). ARK5 protein expression was confirmed by western blot in MM.1R, MM.1S, RPMI-8226, ARP1, EJM and JJN3 and was significantly higher in the more sensitive cell lines EJM and JJN3. Transcriptomic differences between more sensitive cell lines EJM, JJN3, NCI-H929 as compared to the six less sensitive cell lines were culled from RNA-seq and analyzed in different databases Genego, IPA and DAVID. Cell cycle was the top KEGG pathway identified in this analysis and included CDK6, a key target of ON123300. Simultaneously a xenograft mouse model was used to evaluate the therapeutic potential of ON123300 in vivo. ARK5 positive MM.1S myeloma cells (with pcDNA-mCh-luc constitutively expressing luciferase) were injected subcutaneously into SCID mice. Following tumor growth the mice were grouped as- control/untreated (n=5) and ON123300 treated (n = 5). ON123300 (100mg/kg) was administered by IP injection on alternative days and tumor growth monitored using highly sensitive IVIS imaging system. Mice in the treated group responded with 100 fold decrease in bioluminescence intensity (Control- 3.7xE10p/sec/cm2/sr, Treated-3.5xE08p/sec/cm2/sr) in their tumors as compared to untreated control mice. Our results suggest that ON123300 to be a potent inhibitor of tumor growth in vitro and in vivo and could be an effective agent for myeloma treatment especially for patients with aggressive disease driven by cytogenetic changes involving the MAF/ MMSET transcription factors. We expect our studies to expand therapeutic options for patients with this challenging disease. Citation Format: Deepak Perumal, Venu Thirukonda, Zewei Jiang, Violetta V. Leshchenko, Pei-yu Kuo, Samira Shahnaz, Jennifer Rubel, Weijia Zhang, Hearn Jay Cho, M.V. Ramana Reddy, E. Premkumar Reddy, Samir Parekh. Dual targeting of CDK4 and ARK5 using a novel kinase inhibitor ON123300 is effective in vitro and in vivo in Multiple Myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1324. doi:10.1158/1538-7445.AM2014-1324