A Novel Peptide that Disrupts the Lck-IP3R Protein-Protein Interaction Induces Widespread Cell Death in Leukemia and Lymphoma.

There is increasing evidence that the T-cell protein, Lck, is involved in the pathogenesis of chronic lymphocytic leukemia (CLL) as well as other leukemias and lymphomas. We previously discovered that Lck binds to domain 5 of inositol 1,4,5-trisphosphate receptors (IP3R) to regulate Ca2+ homeostasis. Using bioinformatics, we targeted a region within domain 5 of IP3R-1 predicted to facilitate protein-protein interactions (PPIs). We generated a synthetic 21 amino acid peptide, KKRMDLVLELKNNASKLLLAI, which constitutes a domain 5 sub-domain (D5SD) of IP3R-1 that specifically binds Lck via its SH2 domain. With the addition of an HIV-TAT sequence to enable cell permeability of D5SD peptide, we observed wide-spread, Ca2+-dependent, cell killing of hematological cancer cells when the Lck-IP3R PPI was disrupted by TAT-D5SD. All cell lines and primary cells were sensitive to D5SD peptide, but malignant T-cells were less sensitive compared with B-cell or myeloid malignancies. Mining of RNA-seq data showed that LCK was expressed in primary diffuse large B-cell lymphoma (DLBCL) as well as acute myeloid leukemia (AML). In fact, LCK shows a similar pattern of expression as many well-characterized AML oncogenes and is part of a protein interactome that includes FLT3-ITD, Notch-1, and Kit. Consistent with these findings, our data suggest that the Lck-IP3R PPI may protect malignant hematopoietic cells from death. Importantly, TAT-D5SD showed no cytotoxicity in three different non-hematopoietic cell lines; thus its ability to induce cell death appears specific to hematopoietic cells. Together, these data show that a peptide designed to disrupt the Lck-IP3R PPI has a wide range of pre-clinical activity in leukemia and lymphoma.

JAK2V617F-mediated phosphorylation of PRMT5 downregulates its methyltransferase activity and promotes myeloproliferation.

The JAK2V617F constitutively activated tyrosine kinase is found in most patients with myeloproliferative neoplasms. While examining the interaction between JAK2 and PRMT5, an arginine methyltransferase originally identified as JAK-binding protein 1, we found that JAK2V617F (and JAK2K539L) bound PRMT5 more strongly than did wild-type JAK2. These oncogenic kinases also acquired the ability to phosphorylate PRMT5, greatly impairing its ability to methylate its histone substrates, and representing a specific gain-of-function that allows them to regulate chromatin modifications. We readily detected PRMT5 phosphorylation in JAK2V617F-positive patient samples, and when we knocked down PRMT5 in human CD34+ cells using shRNA, we observed increased colony formation and erythroid differentiation. These results indicate that phosphorylation of PRMT5 contributes to the mutant JAK2-induced myeloproliferative phenotype.

Induction of Ca²+-driven apoptosis in chronic lymphocytic leukemia cells by peptide-mediated disruption of Bcl-2-IP3 receptor interaction.

Bcl-2 contributes to the pathophysiology and therapeutic resistance of chronic lymphocytic leukemia (CLL). Therefore, developing inhibitors of this protein based on a thorough understanding of its mechanism of action is an active and promising area of inquiry. One approach centers on agents (eg, ABT-737) that compete with proapoptotic members of the Bcl-2 protein family for binding in the hydrophobic groove formed by the BH1-BH3 domains of Bcl-2. Another region of Bcl-2, the BH4 domain, also contributes to the antiapoptotic activity of Bcl-2 by binding to the inositol 1,4,5-trisphosphate receptor (IP3R) Ca²(+) channel, inhibiting IP(3)-dependent Ca²(+) release from the endoplasmic reticulum. We report that a novel synthetic peptide, modeled after the Bcl-2-interacting site on the IP3R, binds to the BH4 domain of Bcl-2 and functions as a competitive inhibitor of the Bcl-2-IP3R interaction. By disrupting the Bcl-2-IP3R interaction, this peptide induces an IP3R-dependent Ca²(+) elevation in lymphoma and leukemia cell lines and in primary CLL cells. The Ca²(+) elevation evoked by this peptide induces apoptosis in CLL cells, but not in normal peripheral blood lymphocytes, suggesting the involvement of the Bcl-2-IP3R interaction in the molecular mechanism of CLL and indicating the potential merit of targeting this interaction therapeutically.

Apoptosis and autophagy: decoding calcium signals that mediate life or death.

Calcium is a versatile and dynamic 2nd messenger that is essential for the survival of all higher organisms. In cells that undergo activation or excitation, calcium is released from the endoplasmic/sarcoplasmic reticulum to activate calcium-dependent kinases and phosphatases, thereby regulating numerous cellular processes; for example, apoptosis and autophagy. In the case of apoptosis, endogenous ligands or pharmacological agents induce prolonged cytosolic calcium elevation, which in turn leads to cell death. In contrast, there is now evidence that calcium regulates autophagy by several mechanisms, and these may be important for maintaining cell survival. Here we summarize what is known about how calcium regulates these life and death decisions. We pay particular attention to pathways that have been described in lymphocytes and cardiomyocytes, as these systems provide optimal models for understanding calcium signaling in the context of normal cell physiology.

Glucocorticoids downregulate Fyn and inhibit IP(3)-mediated calcium signaling to promote autophagy in T lymphocytes.

T cell receptor activation induces inositol 1,4,5 trisphosphate (IP(3))-mediated calcium signaling that is essential for cell metabolism and survival. Moreover, inhibitors of IP(3) or pharmacological agents that disrupt calcium homeostasis readily induce autophagy. Using a glucocorticoid-sensitive CD4/CD8 positive T cell line, we found that dexamethasone prevented both IP(3)-mediated and spontaneous calcium signals within a timeframe that correlated with the induction of autophagy. We determined that this loss in IP(3)-mediated calcium signaling was dependent upon the downregulation of the Src kinase Fyn at the mRNA and protein level. Because it has previously been shown that Fyn positively regulates IP(3)-mediated calcium release by phosphorylating Type I IP(3) receptors (IP(3)R1), we investigated the effect of glucocorticoids on IP(3)R1 phosphorylation at Tyr353. Accordingly, glucocorticoid-mediated downregulation of Fyn prevented IP(3)R1 phosphorylation at Tyr353. Moreover, selective knockdown of Fyn or treatment with a Src inhibitor also attenuated IP(3)-mediated calcium release and induced autophagy. Collectively, these data indicate that glucocorticoids promote autophagy by inhibiting IP(3)-dependent calcium signals. These findings carry important therapeutic implications given the widespread use of dexamethasone as both a chemotherapeutic and immunosuppressive agent.

Glucocorticoid-mediated inhibition of Lck modulates the pattern of T cell receptor-induced calcium signals by down-regulating inositol 1,4,5-trisphosphate receptors.

Glucocorticoids are potent immunosuppressive agents that block upstream signaling events required for T cell receptor (TCR) activation. However, the mechanism by which glucocorticoids inhibit downstream responses, such as inositol 1,4,5-trisphosphate (IP(3))-induced calcium signals, is not completely understood. Here we demonstrate that low concentrations of dexamethasone rapidly convert transient calcium elevations to oscillations after strong TCR stimulation. Dexamethasone converted the pattern of calcium signaling by inhibiting the Src family kinase Lck, which was shown to interact with and positively regulate Type I IP(3) receptor. In addition, low concentrations of dexamethasone were sufficient to inhibit calcium oscillations and interleukin-2 mRNA after weak TCR stimulation. Together, these findings indicate that by inhibiting Lck and subsequently down-regulating IP(3) receptors, glucocorticoids suppress immune responses by weakening the strength of the TCR signal.

The interactive transcript abundance index [c-myc*p73alpha]/[p21*Bcl-2] correlates with baseline level of apoptosis and response to CPT-11 in human bronchogenic carcinoma cell lines.

Currently available cytotoxic chemotherapy is ineffective for treating bronchogenic carcinoma, and this is partly due to unpredictable inter-tumor variation in resistance. For example, tumors with inactivating p53 mutations or deletions are less likely to respond to certain chemotherapeutics. However, even if p53 is intact, a tumor may be unresponsive if defects in other p53 pathway genes compromise apoptosis. In an effort to identify biomarkers that better predict response to camptothecin, we investigated the association of CPT-11 (irinotecan)-induced cytotoxicity (IC50) with apoptosis or expression of genes upstream or downstream of p53 in cell lines that retain wild-type p53. CPT-11 response was greater in cell lines with higher baseline apoptosis (p<0.05). In addition, the interactive transcript abundance index (ITAI) [c-myc*p73alpha]/[p21*Bcl-2] was directly correlated with baseline apoptosis (p<0.01) and CPT-11 response (p<0.05). The ITAI was also correlated with CPT-11 response among cell lines derived from a variety of tissues that had inactivating p53 mutations or deletions, supporting its applicability for predicting response to camptothecins in other tissues regardless of p53 status.

Stable low-level expression of p21WAF1/CIP1 in A549 human bronchogenic carcinoma cell line-derived clones down-regulates E2F1 mRNA and restores cell proliferation control.

BACKGROUND: Deregulated cell cycle progression and loss of proliferation control are key properties of malignant cells. In previous studies, an interactive transcript abundance index (ITAI) comprising three cell cycle control genes, [MYC x E2F1]/p21 accurately distinguished normal from malignant bronchial epithelial cells (BEC), using a cut-off threshold of 7,000. This cut-off is represented by a line with a slope of 7,000 on a bivariate plot of p21 versus [MYC x E2F1], with malignant BEC above the line and normal BEC below the line. This study was an effort to better quantify, at the transcript abundance level, the difference between normal and malignant BEC. The hypothesis was tested that experimental elevation of p21 in a malignant BEC line would decrease the value of the [MYC x E2F1]/p21 ITAI to a level below this line, resulting in loss of immortality and limited cell population doubling capacity. In order to test the hypothesis, a p21 expression vector was transfected into the A549 human bronchogenic carcinoma cell line, which has low constitutive p21 TA expression relative to normal BEC. RESULTS: Following transfection of p21, four A549/p21 clones with stable two-fold up-regulated p21 expression were isolated and expanded. For each clone, the increase in p21 transcript abundance (TA) was associated with increased total p21 protein level, more than 5-fold reduction in E2F1 TA, and 10-fold reduction in the [MYC x E2F1]/p21 ITAI to a value below the cut-off threshold. These changes in regulation of cell cycle control genes were associated with restoration of cell proliferation control. Specifically, each transfectant was capable of only 15 population doublings compared with unlimited population doublings for parental A549. This change was associated with an approximate 2-fold increase in population doubling time to 38.4 hours (from 22.3 hrs), resumption of contact-inhibition, and reduced dividing cell fraction as measured by flow cytometric DNA analysis. CONCLUSION: These results, likely due to increased p21-mediated down-regulation of E2F1 TA at the G1/S phase transition, are consistent with our hypothesis. Specifically, they provide experimental confirmation that a line with slope of 7,000 on the p21 versus [MYC x E2F1] bivariate plot quantifies the difference between normal and malignant BEC at the level of transcript abundance.

Computer identification of snoRNA genes using a Mammalian Orthologous Intron Database.

Based on comparative genomics, we created a bioinformatic package for computer prediction of small nucleolar RNA (snoRNA) genes in mammalian introns. The core of our approach was the use of the Mammalian Orthologous Intron Database (MOID), which contains all known introns within the human, mouse and rat genomes. Introns from orthologous genes from these three species, that have the same position relative to the reading frame, are grouped in a special orthologous intron table. Our program SNO.pl searches for conserved snoRNA motifs within MOID and reports all cases when characteristic snoRNA-like structures are present in all three orthologous introns of human, mouse and rat sequences. Here we report an example of the SNO.pl usage for searching a particular pattern of conserved C/D-box snoRNA motifs (canonical C- and D-boxes and the 6 nt long terminal stem). In this computer analysis, we detected 57 triplets of snoRNA-like structures in three mammals. Among them were 15 triplets that represented known C/D-box snoRNA genes. Six triplets represented snoRNA genes that had only been partially characterized in the mouse genome. One case represented a novel snoRNA gene, and another three cases, putative snoRNAs. Our programs are publicly available and can be easily adapted and/or modified for searching any conserved motifs within mammalian introns.

Variation in transcriptional regulation of cyclin dependent kinase inhibitor p21waf1/cip1 among human bronchogenic carcinomas.

BACKGROUND: Cell proliferation control depends in part on the carefully ordered regulation of transcription factors. The p53 homolog p73, contributes to this control by directly upregulating the cyclin dependent kinase inhibitor, p21waf1/cip1. E2F1, an inducer of cell proliferation, directly upregulates p73 and in some systems upregulates p21 directly. Because of its central role in controlling cell proliferation, upregulation of p21 has been explored as a modality for treating bronchogenic carcinoma (BC). Improved understanding of p21 transcriptional regulation will facilitate identification of BC tissues that are responsive to p21-directed therapies. Toward this goal, we investigated the role that E2F1 and p73 each play in the transcriptional regulation of p21. RESULTS: Among BC samples (N = 21) p21 transcript abundance (TA) levels varied over two orders of magnitude with values ranging from 400 to 120,000 (in units of molecules/106 molecules beta-actin). The p21 values in many BC were high compared to those observed in normal bronchial epithelial cells (BEC) (N = 18). Among all BC samples, there was no correlation between E2F1 and p21 TA but there was positive correlation between E2F1 and p73alpha (p < 0.001) TA. Among BC cell lines with inactivated p53 and wild type p73 (N = 7) there was positive correlation between p73alpha and p21 TA (p < 0.05). Additionally, in a BC cell line in which both p53 and p73 were inactivated (H1155), E2F1 TA level was high (50,000), but p21 TA level was low (470). Transiently expressed exogenous p73alpha in the BC cell line Calu-1, was associated with a significant (p < 0.05) 90% increase in p21 TA and a 20% reduction in E2F1 TA. siRNA mediated reduction of p73 TA in the N417 BC cell line was associated with a significant reduction in p21 TA level (p < 0.01). CONCLUSION: p21 TA levels vary considerably among BC patients which may be attributable to 1) genetic alterations in Rb and p53 and 2) variation in TA levels of upstream transcription factors E2F1 and p73. Here we provide evidence that p73 upregulates p21 TA in BC tissues and upregulated p21 TA may result from E2F1 upregulation of p73 but not from E2F1 directly.

The c-myc x E2F-1/p21 interactive gene expression index augments cytomorphologic diagnosis of lung cancer in fine-needle aspirate specimens.

Morphological analysis of cytologic samples obtained by fine-needle aspirate (FNA) or bronchoscopy is an important method for diagnosing bronchogenic carcinoma. However, this approach has only about 65 to 80% diagnostic sensitivity. Based on previous studies, the c-myc x E2F-1/p21WAF1/CIP1 (p21 hereafter) gene expression index is highly sensitive and specific for distinguishing normal from malignant bronchial epithelial tissues. In an effort to improve sensitivity of diagnosing lung cancer in cytologic specimens, we used Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR) to measure the c-myc x E2F-1/p21 index in cDNA samples from 14 normal lung samples (6 normal lung parenchyma and 8 normal bronchial epithelial cell [NBEC] biopsies), and 16 FNA biopsies from 14 suspected tumors. Based on cytomorphologic criteria, 11 of the 14 suspected tumors were diagnosed as bronchogenic carcinoma and three specimens were non-diagnostic. Subsequent biopsy samples confirmed that the three non-diagnostic samples were derived from lung carcinomas. The index value for each bronchogenic carcinoma was above a cut-off value of 7000 and the index value of all but one normal sample was below 7000. Thus the c-myc x E2F-1/p21 index may augment cytomorphologic diagnosis of bronchogenic carcinoma biopsy samples, particularly those considered non-diagnostic by cytomorphologic criteria.