Jason J. Pitt

Inducing apoptosis in chemotherapy‐resistant B‐lineage acute lymphoblastic leukaemia cells by targeting HSPA5, a master regulator of the anti‐apoptotic unfolded protein response signalling network

We present previously unknown evidence that the immunoglobulin heavy chain binding protein BIP/HSPA5, also known as glucose regulated protein (GRP)78, serving as a pivotal component of the pro‐survival axis of the unfolded protein response (UPR) signalling network, is abundantly expressed in relapsed B‐lineage acute lymphoblastic leukaemia (ALL) and contributes to chemotherapy resistance of leukaemic B‐cell precursors. The resistance of B‐lineage ALL cells to the standard anti‐leukaemic drug vincristine was overcome by the HSPA5 inhibitor epigallocatechin gallate, which inhibits the anti‐apoptotic function of HSPA5 by targeting its ATP‐binding domain. Notably, chemotherapy‐resistant B‐lineage ALL cells underwent apoptosis within 48 h of exposure to a doxorubicin‐conjugated cell‐penetrating cyclic anti‐HSPA5 peptide targeting surface‐expressed HSPA5 molecules on leukaemia cells. The identification of the HSPA5 as a chemoresistance biomarker and molecular target for B‐lineage ALL may lead to new anti‐leukaemic treatment strategies that are much needed.

Systems-wide RNAi analysis of CASP8AP2 / FLASH shows transcriptional deregulation of the replication-dependent histone genes and extensive effects on the transcriptome of colorectal cancer cells

BackgroundColorectal carcinomas (CRC) carry massive genetic and transcriptional alterations that influence multiple cellular pathways. The study of proteins whose loss-of-function (LOF) alters the growth of CRC cells can be used to further understand the cellular processes cancer cells depend upon for survival.ResultsA small-scale RNAi screen of ~400 genes conducted in SW480 CRC cells identified several candidate genes as required for the viability of CRC cells, most prominently CASP8AP2/FLASH. To understand the function of this gene in maintaining the viability of CRC cells in an unbiased manner, we generated gene specific expression profiles following RNAi. Silencing of CASP8AP2/FLASH resulted in altered expression of over 2500 genes enriched for genes associated with cellular growth and proliferation. Loss of CASP8AP2/FLASH function was significantly associated with altered transcription of the genes encoding the replication-dependent histone proteins as a result of the expression of the non-canonical polyA variants of these transcripts. Silencing of CASP8AP2/FLASH also mediated enrichment of changes in the expression of targets of the NFκB and MYC transcription factors. These findings were confirmed by whole transcriptome analysis of CASP8AP2/FLASH silenced cells at multiple time points. Finally, we identified and validated that CASP8AP2/FLASH LOF increases the expression of neurofilament heavy polypeptide (NEFH), a protein recently linked to regulation of the AKT1/ß-catenin pathway.ConclusionsWe have used unbiased RNAi based approaches to identify and characterize the function of CASP8AP2/FLASH, a protein not previously reported as required for cell survival. This study further defines the role CASP8AP2/FLASH plays in the regulating expression of the replication-dependent histones and shows that its LOF results in broad and reproducible effects on the transcriptome of colorectal cancer cells including the induction of expression of the recently described tumor suppressor gene NEFH.

Genetic Amplification of the NOTCH Modulator LNX2 Upregulates the WNT/β-Catenin Pathway in Colorectal Cancer

Chromosomal copy number alterations (aneuploidy) define the genomic landscape of most cancer cells, but identification of the oncogenic drivers behind these imbalances remains an unfinished task. In this study, we conducted a systematic analysis of colorectal carcinomas that integrated genomic copy number changes and gene expression profiles. This analysis revealed 44 highly overexpressed genes mapping to localized amplicons on chromosome 13, gains of which occur often in colorectal cancers (CRC). RNA interference (RNAi)-mediated silencing identified eight candidates whose loss-of-function reduced cell viability 20% or more in CRC cell lines. The functional space of the genes NUPL1, LNX2, POLR1D, POMP, SLC7A1, DIS3, KLF5, and GPR180 was established by global expression profiling after RNAi exposure. One candidate, LNX2, not previously known as an oncogene, was involved in regulating NOTCH signaling. Silencing LNX2 reduced NOTCH levels but also downregulated the transcription factor TCF7L2 and markedly reduced WNT signaling. LNX2 overexpression and chromosome 13 amplification therefore constitutively activates the WNT pathway, offering evidence of an aberrant NOTCH-WNT axis in CRC.

Identification of novel molecular regulators of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in breast cancer cells by RNAi screening

IntroductionTumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to its receptors, TRAIL-receptor 1 (TRAIL-R1) and TRAIL-receptor 2 (TRAIL-R2), leading to apoptosis by activation of caspase-8 and the downstream executioner caspases, caspase-3 and caspase-7 (caspase-3/7). Triple-negative breast cancer (TNBC) cell lines with a mesenchymal phenotype are sensitive to TRAIL, whereas other breast cancer cell lines are resistant. The underlying mechanisms that control TRAIL sensitivity in breast cancer cells are not well understood. Here, we performed small interfering RNA (siRNA) screens to identify molecular regulators of the TRAIL pathway in breast cancer cells.MethodsWe conducted siRNA screens of the human kinome (691 genes), phosphatome (320 genes), and about 300 additional genes in the mesenchymal TNBC cell line MB231. Forty-eight hours after transfection of siRNA, parallel screens measuring caspase-8 activity, caspase-3/7 activity, or cell viability were conducted in the absence or presence of TRAIL for each siRNA, relative to a negative control siRNA (siNeg). A subset of genes was screened in cell lines representing epithelial TNBC (MB468), HER2-amplified breast cancer (SKBR3), and estrogen receptor-positive breast cancer (T47D). Selected putative negative regulators of the TRAIL pathway were studied by using small-molecule inhibitors.ResultsThe primary screens in MB231 identified 150 genes, including 83 kinases, 4 phosphatases, and 63 nonkinases, as potential negative regulators of TRAIL. The identified genes are involved in many critical cell processes, including apoptosis, growth factor-receptor signaling, cell-cycle regulation, transcriptional regulation, and DNA repair. Gene-network analysis identified four genes (PDPK1, IKBKB, SRC, and BCL2L1) that formed key nodes within the interaction network of negative regulators. A secondary screen of a subset of the genes identified in additional cell lines representing different breast cancer subtypes and sensitivities to TRAIL validated and extended these findings. Further, we confirmed that small-molecule inhibition of SRC or BCL2L1, in combination with TRAIL, sensitizes breast cancer cells to TRAIL-induced apoptosis, including cell lines resistant to TRAIL-induced cytotoxicity.ConclusionsThese data identify novel molecular regulators of TRAIL-induced apoptosis in breast cancer cells and suggest strategies for the enhanced application of TRAIL as a therapy for breast cancer.

Loss-of-function RNAi screens in breast cancer cells identify AURKB, PLK1, PIK3R1, MAPK12, PRKD2, and PTK6 as sensitizing targets of rapamycin activity.

The use of molecularly targeted drugs as single agents has shown limited utility in many tumor types, largely due to the complex and redundant nature of oncogenic signaling networks. Targeting of the PI3K/AKT/mTOR pathway through inhibition of mTOR in combination with aromatase inhibitors has seen success in particular sub-types of breast cancer and there is a need to identify additional synergistic combinations to maximize the clinical potential of mTOR inhibitors. We have used loss-of-function RNAi screens of the mTOR inhibitor rapamycin to identify sensitizers of mTOR inhibition. RNAi screens conducted in combination with rapamycin in multiple breast cancer cell lines identified six genes, AURKB, PLK1, PIK3R1, MAPK12, PRKD2, and PTK6 that when silenced, each enhanced the sensitivity of multiple breast cancer lines to rapamycin. Using selective pharmacological agents we confirmed that inhibition of AURKB or PLK1 synergizes with rapamycin. Compound-associated gene expression data suggested histone deacetylation (HDAC) inhibition as a strategy for reducing the expression of several of the rapamycin-sensitizing genes, and we tested and validated this using the HDAC inhibitor entinostat in vitro and in vivo. Our findings indicate new approaches for enhancing the efficacy of rapamycin including the use of combining its application with HDAC inhibition.

JAK3 pathway is constitutively active in B-lineage acute lymphoblastic leukemia

In this article, we report that primary leukemic B-cell precursors from B-lineage acute lymphoblastic leukemia (ALL) patients overexpress multiple JAK3-activating cytokines as well as their receptors. We also show that amplified expression of JAK3 pathway genes in B-lineage ALL is associated with steroid resistance and relapse. Our findings further demonstrate that several different diagnostic classes of B-lineage lymphoid malignancies exhibit upregulated expression of JAK3 pathway genes, which are associated with an overexpression of genes for JAK3-stimulatory cytokines with concomitant deficiency of JAK3-inhibitory signaling molecules. Thus, despite the rare occurrence of activating JAK3 mutations, JAK3 appears to be constitutively active and represents a viable molecular target in the treatment of a broad range of B-lineage lymphoid malignancies, including B-lineage ALL.

Genomic instability and mouse microRNAs

Tumor progression is the continual selection of variant subpopulations of malignant cells that have acquired increasing levels of genetic instability (Nowell Science 1976, 194, 23–28). This instability is manifested as chromosomal aneuploidy or translocations, viral integration or somatic mutations that typically affect the expression of a gene (oncogene) that is especially damaging to the proper function of a cell. With the recent discovery of non-coding RNAs such as microRNAs (miRNAs), the concept that a target of genetic instability must be a protein-encoding gene is no longer tenable. Over the years, we have conducted several studies comparing the location of miRNA genes to positions of genetic instability, principally retroviral integration sites and chromosomal translocations in the mouse as a means of identifying miRNAs of importance in carcinogenesis. In this current study, we have used the most recent annotation of the mouse miRome (miRBase, release 16.0), and several datasets reporting the sites of integration of different retroviral vectors in a variety of mouse strains and mouse models of cancer, including for the first time a model that shows a propensity to form solid tumors, as a means to further identify or define, candidate oncogenic miRNAs. Several miRNA genes and miRNA gene clusters stand out as interesting new candidate oncogenes due to their close proximity to common retroviral integration sites including miR-29a/b/c and miR106a~363. We also discussed some recently identified miRNAs including miR-1965, miR-1900, miR-1945, miR-1931, miR-1894, and miR-1936 that are close to common retroviral integration sites and are therefore likely to have some role in cell homeostasis.

Abstract 1952: The rapid generation of mouse B cell lymphomas by lentiviral mediated overexpression of miR-1204 from a genetically unstable region of human 8q24

SNPs in the area surrounding MYC on human 8q24 have recently been found by Genome Wide Association (GWA) to be associated with susceptibility to a number of malignancies including prostate, breast, colorectal and bladder carcinomas (CA). This genetically unstable region is also a frequent target of chromosomal translocation (Tx), amplification or retroviral integration in a number of CAs such as breast, prostate, ovarian, colon, pancreatic, and cervical. In one example, essentially 100% of patients with Burkitt9s lymphoma exhibit one of three characteristic non-random chromosomal Txs that places MYC or the immediate surrounding region in close proximity to enhancers of the immunoglobulin (Ig) heavy chain or light chain loci. Although de-regulated MYC expression could be assumed to be the target of the genomic instability or GWA-based susceptibility, no clear correlation between MYC expression and disease has been established. A possible alternative target has been identified in a series of transcripts cloned from the PVT1 region downstream of MYC. However, the extent of alternative splicing coupled with the lack of a coding region has made it difficult to assign a specific role for any PVT1-derived transcripts. Recently, we have identified a cluster of small RNAs exhibiting the hairpin formation, sequence conservation and expression characteristics of miRNAs (miR-1204∼1208) within the transcriptional domain of PVT1. Increased expression of several of these miRNAs in tumors harboring amplified MYC/PVT1 or Burkitt lymphomas with the 8q24 Tx suggests a possible role for these miRNAs in tumorigenesis, especially for miR-1204 which is found 60 kb downstream of MYC, flanking exon 1b of PVT1. An increased expression of miR-1204 in pre-B cells compared to pro-B cells also suggested a lymphoid specific developmental pattern of expression. Lentiviral constructs of miR-1204 under control of a CMV promoter (LentiCMV-miR-1204) revealed a possible effect on MYC, but in a pre-B (not pro-B) specific environment. Introduction of LentiCMV-miR-1204 into mice harboring either a MYC (C.iMYC) or IL6 (C.IL6) transgene resulted in high frequency and rapid onset of large B cell lymphomas (∼16 or 82 days vs. 91 or 117 days for controls, respectively). While these results point to over-expression of miR-1204 playing a specific role in the development of B cell malignancy, additional studies with the LentiCMV-miR-1204 construct in other tumor cell lines (prostate and breast) also reveal phenotypic changes in cell proliferation and migration. Further studies using lentiviruses with alternative promoters for miR-1204 and the other 8q24 associated miRNAs are underway to identify downstream targets and pathways not only in lymphoid malignancy but also in the wide range of malignancies associated with GWA-susceptibility and genomic instability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1952.

Abstract 1834: Noncoding RNAs of the 8q24 locus: Consequences of the over-expression or suppression of miR-1204 and PVT1 in developing B cells.

Immortalized models of the mouse B lymphocyte lineage have proven to be valuable resources for a wide variety of B cell neoplasms and for the study of normal B cell differentiation. Whether these models have been engineered in mice (in vivo) or in cell culture (in vitro), there are distinct advantages and disadvantages to both. Homogeneity and ease of accessibility can be considered a benefit of working with cultured cells, whereas the contribution from accessory or stromal cells can be an important element not readily available in the in vitro model system. We originally used immortalized Burkitt9s Lymphoma (BL) cells to discover a cluster of microRNAs, miR-1204∼1208, that map within the noncoding PVT1 locus in the region of human 8q24 region down-stream of MYC. The BL cell lines allowed us to isolate a large amount of RNA from a homogeneous resource that enhanced discovery of low-level transcripts. Using probes for mouse miR-1204∼1208 to examine expression in a panel of mouse cell lines representing different stages of B cell development, we were able to show that expression of miR-1204∼1208 appeared to arise at the small B cell stage and that these higher levels of expression continued through to the mature plasma cell. This suggested that the pre-B cell or naive small B cell stages may be most illuminating in assigning targets and function to miR-1204∼1208 or PVT1. To determine if over-expression of one of these miRNAs, miR-1204, influences the latency and/or type of B cell malignancy we used two mouse transgenic (TG) models of B cell malignancy, H2-L d -hu-IL6 and iMyc, lentiviral expression of miR-1204 reduced the latency of tumor development in both models and in the case of iMyc-TG, there was also a shift in tumor type from late stage plasmacytoma to the earlier stage of large B cell lymphoma. However, further interpretation of these results was confounded by heterogeneity of lentiviral integration and expression among tumors. Thus, we turned to several human and mouse in vitro models of B cell development, representing pro-B, pre-B, small B, mature B and plasma cell stages to address the effects of modulating the expression of miR-1204 and its host noncoding transcript, PVT1. Over-expression of miR-1204 has been achieved through the use of lentiviruses or synthetic mimics and suppressed expression has been achieved through application of synthetic inhibitors. We also over-expressed PVT1 using a lentiviral vector and suppressed PVT1 expression through the use of siRNA corresponding to various exons of PVT1. Resultant changes in growth and morphology of these cell lines hint that microarray expression analyses will reveal functional targets of miR-1204 in normal and malignant lymphoid development. It will also be of interest to examine whether over-expression or inhibition of miR-1204 plays an additional role in maturation of the normal B cell. Citation Format: Konrad Huppi, Oliver L. Ou, Jason J. Pitt, Brady Wahlberg, Tamara L. Jones, Vishala Neppalli, Siegfried Janz, Natasha J. Caplen. Noncoding RNAs of the 8q24 locus: Consequences of the over-expression or suppression of miR-1204 and PVT1 in developing B cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1834. doi:10.1158/1538-7445.AM2013-1834

Abstract 199: Noncoding RNAs of the 8q24 locus

The 8q24 locus has been found to be involved in many types of cancers as a consequence of somatic changes associated with chromosome instability including amplification, translocation or deletion or frequent viral integration (HPV). A number of SNPs in Genome Wide Association (GWA) studies have also implicated the 8q24 locus as a region of susceptibility for many types of cancer. The most likely 8q24 candidate target may be the MYC proto-oncogene that is a well characterized transcription factor. However, the assumed correlation between MYC expression and disease is lacking suggesting a connection between 8q24 involvement and disease is much more complicated than simply targeting MYC. While other transcription units also reside within the 8q24 locus (PRNCR1, POU5F1P1, PVT1 and the miRNA cluster miR-1204∼1208), they are remarkable in that no coding potential has been readily associated with any of these genes. Thus, the region has been referred to as the “8q24 Gene Desert”. With the renewed realization that many noncoding RNAs do have a functional role, the location of the miR-1204∼1208 cluster of miRNAs within the PVT1 lincRNA transcriptional unit actually suggests an “Oasis of transcription” that could be the additional or alternative target to MYC. We have now compared expression of transcripts of the miR-1204∼1208 cluster with MYC and PVT1 in multiple cancer cell lines and we have found them to be uniformly up-regulated in expression, particularly in those cell lines with amplified or translocated 8q24. These results confirm a pilot study of microdissected primary prostate tumors that also show correlated high expression in MYC, miR-1204∼1208 and PVT1. To further analyze the functional role of the 8q24 transcripts, we have now introduced mimics or inhibitors of each miRNA and siRNAs corresponding to MYC and PVT1 into prostate or colon cell lines (with and without amplified 8q24). In amplified prostate cell lines, mimics of miR-1204, miR-1206 and miR-1208 appear to be as effective as MYC or PVT1 siRNAs in arresting cell growth and inducing apoptosis. This result is not seen in normal prostate cells. Predictably, inhibitors of the miR-1204, miR-1206 and miR-1208 seem to have no effect or seem to enhance cellular proliferation. In colon cell lines with amplified 8q24, miR-1204, miR-1206, miR-1207 and miR-1208 mimics all appear to be as effective in arresting growth as silencing MYC or PVT1. What is particularly striking is that down-regulation or silencing of PVT1 produces the same effect as over-expression (mimic) of the embedded cluster of miRNAs suggesting opposing actions of miRNAs and host transcript. Experiments to determine whether synergy or promoter competition of the PVT1/miR-1204∼1208 cluster is specifically active in amplified 8q24 will be presented. Nevertheless, we can already assign viable functional roles to transcripts besides MYC that could represent alternative molecular targets within the 8q24 locus for cancer susceptibility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 199. doi:1538-7445.AM2012-199