Developmental regulation of mitochondrial apoptosis by c-Myc governs age- and tissue-specific sensitivity to cancer therapeutics

It is not understood why healthy tissues can exhibit varying levels of sensitivity to the same toxic stimuli. Using BH3 profiling, we find that mitochondria of many adult somatic tissues, including brain, heart, and kidneys, are profoundly refractory to pro-apoptotic signaling, leading to cellular resistance to cytotoxic chemotherapies and ionizing radiation. In contrast, mitochondria from these tissues in young mice and humans are primed for apoptosis, predisposing them to undergo cell death in response to genotoxic damage. While expression of the apoptotic protein machinery is nearly absent by adulthood, in young tissues its expression is driven by c-Myc, linking developmental growth to cell death. These differences may explain why pediatric cancer patients have a higher risk of developing treatment-associated toxicities

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Interleukin-21 Induces Apoptosis of Diffuse Large B Cell Lymphomas via Activation of the STAT3 - c-Myc Intracellular Signaling Pathway

Interleukin-21 (IL-21), a recently discovered member of the IL-2 cytokine family, has been shown to have diverse regulatory effects on B cells including the induction of antibody secretion, differentiation, or apoptosis depending on the cell milieu and activation status. However, the effects of IL-21 on B cell neoplasms such as diffuse large B cell lymphoma (DLBCL) are largely unknown. Our research uncovered the widespread expression of the IL-21 receptor (IL-21R) in B cell lymphomas including DLBCL and that IL-21 stimulation resulted in potent phosphorylation of STAT1 and 3 and weak activation of STAT5. However, our findings also showed that treatment of DLBCL cell lines with IL-21 induced cell cycle arrest and apoptosis. The cell death was caspase-dependent and evident in a majority of DLBCL cell lines. To further examine the potential therapeutic applicability of IL-21, we assessed the effects of IL-21 on primary DLBCL tumors and in vivo DLBCL xenografts in mice. In primary tumors, IL-21 induced apoptosis in five of five DLBCLs compared to two of three follicular lymphomas and two of seven chronic lymphocytic leukemias. No apoptosis or cell death was induced in normal peripheral B lymphocytes. In mice bearing DLBCL xenograft tumors, in situ IL-21 injections induced tumor regression and dramatically extended the overall survival of mice (P\u3c0.001). To elucidate the mechanism of IL-21-induced cell death we analyzed the expression of apoptosis-regulating proteins and observed a strong downregulation of anti-apoptotic Bcl-2 and Bcl-XL and an upregulation of pro-apoptotic Bax post IL-21 stimulation. Subsequent experiments showed that ectopic expression of Bcl-2 or Bcl-XL was able to partially reduce cell death induced by IL-21 while Bax knockdown with siRNA blocked apoptosis completely. To gain insight into the signaling pathways shifting the expression of these proteins toward cell death we performed microarray analysis on sensitive and resistant DLBCL cell lines. The most striking difference in gene expression was observed in C-MYC which was only induced in cell lines exhibiting apoptosis upon IL-21 treatment. Previous reports have shown that c-Myc, which has been studied extensively for its oncogenic properties, can induce apoptosis via downregulation of its transcriptional targets Bcl-2 and Bcl-XL. We then showed that IL-21-induced cell death is dependent on c-Myc by utilizing specific siRNA and shRNA to block the upregulation of this transcription factor and prevent apoptosis. Since c-Myc is a bona-fide target of STAT3 we also showed that siRNA-mediated knockdown of STAT3 abrogated apoptosis by preventing c-Myc upregulation and its subsequent effects on apoptosis-regulating proteins. Our results delineate a novel IL-21 pro-apoptotic signaling pathway and one of the first examples in which the STAT3 - c-Myc pathway, which usually promotes B cell survival and oncogenesis, can be exploited for treatment of cancer. Furthermore, our findings demonstrate that IL-21 is a highly potent anti-DLBCL agent in vitro and in animal models and should be examined in clinical studies of DLBCL

Interleukin 21 - its potential role in the therapy of B-cell lymphomas

Interleukin-21 (IL-21), a member of IL-2 cytokine family, has pleotropic biological effects on lymphoid and myeloid cells. During the past 15 years, since the discovery of IL-21, great advances have been made regarding its biological activity and the mechanisms controlling IL-21-mediated cellular responses, especially in hematological malignancies. Preclinical studies have shown that IL-21R is expressed on healthy and neoplastic B-cells and exogenous IL-21 can induce direct apoptosis of IL-21R expressing B-cell non-Hodgkin lymphomas (NHL), making it a potentially attractive anti-lymphoma therapy. However, in some hematological malignancies such as multiple myeloma, Hodgkin lymphoma and Burkitt lymphoma, IL-21 can induce proliferation of neoplastic B-cells. In NHL, the underlying mechanism of cell death was found to be different between the various subtypes, including activation of different JAK/STAT signal transduction pathways or other factors. Immunomodulatory effects of IL-21 have also been reported to contribute to its anti-tumor effects as described by earlier studies in solid tumors and B-cell associated malignancies. These effects are predominantly mediated by IL-21's ability to activate cytolytic activities by NK-cells and CD4 + /CD8 + T-cells. In this review, we provide an overview of IL-21's effects in NHL, results from clinical trials utilizing IL-21, and propose how IL-21 can be therapeutically exploited for treating these lymphomas

Abstract LB-328: Preclinical activity of interleukin 21 in mantle cell lymphoma

Abstract Mantle cell lymphoma (MCL) is a distinct subtype of non-Hodgkin lymphoma characterized by overexpression of cyclin D1 (CCND1) in 95% of patients due to the t(11;14)(q13;q32) chromosomal translocation. This incurable lymphoma is highly chemoresistant with short duration response, frequent relapses and eventual death, even with the most aggressive chemotherapeutic regimens. Interleukin 21 (IL21), a member of the IL2 cytokine family, has shown anti-tumor effects in solid tumors, chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL) and it is currently in clinical trials for renal cell carcinoma and metastatic melanoma. Herein, we carried out a comprehensive study to delineate the effects of IL21 on MCL cell lines and primary tumors. Flow-cytometric analysis revealed that all MCL cell lines (Mino, HBL-2, Jeko-1, G-519, IRM-2, SP53, Z138, UPN1 and L-128) as well as primary tumors expressed surface IL21R at variable levels. Treatment of Mino, HBL-2 and SP53 cells with IL21 (100ng/mL) led to a marked time-dependent decrease in cell proliferation and increased cell death. In contrast, Jeko1, IRM2, L128, Z138, UPN1 and G519 cells exhibited resistance to IL21 treatment. Similarly, primary MCL tumors treated with IL21 in vitro exhibited significant cell death in 3 of 5 cases. To decipher the mechanism of IL21-induced apoptosis, responsive and resistant cell lines as well as primary tumors were utilized. Similarly to our previous study in DLBCL, IL21 stimulation resulted in dramatic phosphorylation of STAT1 and STAT3 in IL-21 responsive cell lines (Mino, HBL-2, SP53) and a primary tumor, while minimal phosphorylation of STAT5 was observed only in Mino. However, observed levels of phosphorylated STAT1, 3 and 5 were significantly lower in the resistant cells (Jeko-1) and primary tumor. We have previously demonstrated that IL21-induced cell death in DLBCL is mediated by STAT3-induced upregulation of c-Myc expression. Correspondingly, IL21 led to c-Myc upregulation in IL21-sensitive MCL cell lines and primary tumors. Strikingly, IL21 failed to induce c-Myc in the resistant cell line and primary tumor despite expressing similar level of IL21R. IL21 treatment also resulted in upregulation of the pro-apoptotic protein Bax and downregulation of the anti-apoptotic proteins Bcl-XL and Bcl2, as previously observed in DLBCL. Moreover, knockdown of STAT3, c-Myc or Bax using specific siRNAs in Mino cells resulted in abrogation of the IL21-induced cell death. In contrast to a previous report, knockdown of STAT1 did not prevent IL21-induced Mino cell death. Overall, these observations suggest that in vitro IL21 induces cell death and apoptosis in a subset of MCL cell lines and primary tumors by activating the STAT3-cMyc pathway and not through the STAT1 signaling pathway. Additionally, from our observations, MCL resistance to IL21 may be due to abolition of STAT3-c-Myc activation in response to IL21. Citation Format: Shruti Bhatt, Kristopher A. Sarosiek, Izidore S. Lossos. Preclinical activity of interleukin 21 in mantle cell lymphoma. [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 LB-328. doi:10.1158/1538-7445.AM2013-LB-32

Interleukin-21 Induces Cell Cycle Arrest and Apoptosis of Diffuse Large B-Cell Lymphomas (DLBCL) Via Activation of STAT3 and Upregulation of C-Myc

Abstract IL-21, a recently discovered member of the IL-2 cytokine family, has been shown to have diverse regulatory effects on B-cells including the induction of antibody secretion, differentiation, or apoptosis depending on the cellular milieu and activation status. However, the effects of IL-21 on B-cell neoplasms such as DLBCL are largely unknown. Our research has uncovered the widespread expression of the IL-21 receptor (IL-21R) in B-cell lymphomas including DLBCL. Our results confirmed reports that IL-21R stimulation results in potent phosphorylation of STAT-1 and -3 and weak activation of STAT-5. However, our findings also show that treatment of DLBCL cell lines with IL-21 induces cell cycle arrest and apoptosis. The cell death is caspase-dependent and evident in a majority of DLBCL cell lines. To further examine the potential therapeutic applicability of IL-21, we assessed the effects of IL-21 on primary DLBCL tumor cells and in vivo DLBCL mice models. In primary tumors, IL-21 induced apoptosis in five of five DLBCLs compared to two of three follicular lymphomas, and two of seven chronic lymphocytic leukemias. No apoptosis or cell death was induced in normal peripheral B-lymphocytes. In xenograft DLBCL tumors, in situ IL-21 injections induced tumor regression and dramatically extended the overall survival of mice (P<0.001). To elucidate the mechanism of IL-21-induced cell death, microarray analysis was performed on endogenously sensitive or resistant DLBCL cell lines as well as an RCK8 cell line that acquired IL-21 resistance following chronic exposure to the cytokine. The most striking difference between IL-21 sensitive and resistant cell lines was observed in the expression of c-Myc, a known target of STAT-3, which was only induced in cell lines undergoing apoptosis upon IL-21 treatment. We have shown that the cell death associated with IL-21 treatment was prevented by utilizing c-Myc specific siRNAs and shRNA. Interestingly, c-Myc upregulation and cell death was also prevented upon knockdown of STAT-3 with siRNAs. Stimulation of the STAT-3-cMYC signaling pathway led to decreases in levels of anti-apoptotic proteins Bcl-XL and Bcl-2, which are bone-fide targets of c-Myc. Our results delineate a new IL-21-induced pro-apoptotic signaling pathway involving STAT-3 and c-Myc, which are usually considered as anti-apoptotic effectors in cancer cells. Furthermore, our findings demonstrate that IL-21 is a highly potent anti-DLBCL agent in vitro and in xenograft DLBCL models and warrant initiation of clinical studies in patients

Interleukin-4 distinctively modifies responses of germinal centre-like and activated B-cell-like diffuse large B-cell lymphomas to immuno-chemotherapy

Diffuse large B-cell lymphomas (DLBCLs) can be classified into two subtypes: germinal-centre B-cell (GCB)-like and Activated B-cell (ABC)-like tumours, which are associated with longer or shorter patient overall survival, respectively. In our previous studies, we have shown that, although DLBCL tumours of GCB-like and ABC-like subtypes express similar levels of IL4 mRNA, they exhibit distinct patterns of IL-4-induced intracellular signalling and different expression of IL-4 target genes. We hypothesized that these differences may contribute to the different clinical behaviour and outcome of DLBCL subtypes. Herein, we demonstrated that IL-4 increased the sensitivity of GCB-like DLBCL to doxorubicin-induced apoptosis and complement-dependent rituximab cell killing. In contrast, IL-4 protected ABC-like DLBCL from the cytotoxic effects of doxorubicin and rituximab. The distinct effects of IL-4 on doxorubicin sensitivity in GCB-like and ABC-like DLBCL cells may be partially attributed to the contrasting effects of the cytokine on Bcl-2 and Bad protein levels in the DLBCL subtypes. These findings suggest that the different effects of IL-4 on chemotherapy and immunotherapy-induced cytotoxicity of GCB- and ABC-like DLBCL could contribute to the different clinical outcomes exhibited by patients with these two subtypes of DLBCL

Differentiation-Stage-Specific Expression of MicroRNAs in B-Lymphocytes and Diffuse Large B-Cell Lymphomas (DLBCL)

Abstract B-cell development and differentiation are complex processes controlled by distinct programs of transcriptional control. A large set of transcriptional factors together or in succession control this process and their deregulation may result in block of differentiation or malignant transformation. MicroRNAs are small RNAs that orchestrate cellular functions by modulating the level of their targeted proteins by either translational arrest or transcript degradation, and play a key role in cell differentiation, apoptosis, proliferation and cancer development. An increasing number of transcription factors are being found targeted by microRNAs. Emerging evidence suggests that differentiation stage-specific expression of microRNAs occurs in the hematopoietic system and during T cell differentiation. Only limited information exists on microRNA expression in normal B cell differentiation and its malignant counterparts. Herein we analyzed microRNA expression profiles in distinct peripheral B cell differentiation stages-naïve, germinal center (GC) centroblasts and memory cells as well as tonsilar T cells. Furthermore, microRNA profiling was performed in germinal center-like (GCB-like) and activated B-cell-like (ABC-like) DLBCL cell lines originating from distinct B-cell differentiation stages. RNA, extracted with mirVana kit (AMBION) from B cell subsets and T cells enriched from normal tonsils was hybridized on LC Sciences (Houston, TX) microarrays harboring 470 human microRNAs probes (Sanger miRBase Release 9.1). Expression of selected microRNAs was confirmed by ABI RT-PCR methodology. Unsupervised clustering of microRNAs with values present in at least 50% of the samples (122 probes) resulted in perfect differentiation-stage clustering of samples. Application of Statistical Analysis of Microarrays (SAM) and Prediction Analysis of Microarrays (PAM) methods (FDR= 10%) identified a 47 microRNA cell of origin classifier for B-cells differentiation stage; 27 of these microRNAs were upregulated and 20 downregulated in centroblasts compared to memory B-cells. MicroRNAs belonging to paralog microRNA clusters (e.g. miR17-92-1, miR363-106a and miR25-106b) demonstrated similar patterns of expression in specific differentiation stages. To identify specific microRNA targets, miRanda, TargetScan and PicTar programs were used. To experimentally confirm the targets, we assessed the effects of specific microRNAs on the expression levels of targeted proteins and on the luciferase reporter under the control of the wild type and mutated 3′ UTR regions of putative target genes. Using this experimental approach we identified lymphocyte-stage-specific microRNAs which expression inversely correlated and might regulate the expression of LMO2, BLIMP1 and IRF4 proteins distinctively expressed at different differentiation stages of B lymphocytes. For example, miR223, which expression is low in GC cells but is high in naïve and memory B cells, downregulates the expression of LMO2. We next analyzed microRNA expression in DLBCL cell lines. Clustering analysis, using the 47 microRNA cell of origin classifier perfectly classified GCB-like and ABC-like cell lines. Interestingly, the expression of microRNAs in both GCB-like and ABC-like DLBCL cell lines was more similar to normal centroblasts than to memory B cells, suggesting that both may originate from distinct subpopulations of GC lymphocytes. The similarity of microRNA expression in cell lines to centroblasts was striking, with only 16 microRNAs (1 upregulated and 15 downregulated in cell lines) showing noticeable differences in levels of expression compared to normal cells. These microRNAs might be involved in the process of lymphoma transformation. SAM analysis aimed to differentiate GCB-like and ABC-like cell lines identified 11 microRNAs, only 3 of which were present in the cell of origin classifier. This observation suggests that there is also a difference in expression of microRNAs not directly related to the distinct cell of origin between the DLBCL subtypes. In summary, our results demonstrate that the microRNA profile changes during the GC reaction as well as during malignant transformation. Specific microRNAs can regulate key transcription factors controlling the processes of lymphocyte differentiation and transformation

Interleukin-21-Induced Apoptosis and Cell Death of Diffuse Large B-Cell Lymphoma (DLBCL) Cell Lines and Primary Tumors Are Associated with an Induction of Bim

Abstract IL-21, a member of the IL-2 cytokine family, is reported to have an immune-mediated anti-tumor activity against renal cell carcinoma, malignant melanoma and Non-Hodgkin’s Lymphoma (NHL) cell lines in xenograph animal models. Whether IL-21 exhibits direct anti-tumor activity against NHL cell lines and primary tumors is presently unknown. We analyzed seven DLBCL and two Burkitt’s lymphoma cell lines for IL-21 receptor (IL-21R) expression. IL-21R was expressed at high levels in the two Burkitt’s lymphoma cell lines (RAJI and RAMOS). Out of the seven DLBCL lines, four expressed high levels of the receptor (OCI-LY-3, OCI-LY-7, OCI-LY-19, and RCK-8) while three had low receptor expression levels (OCI-LY-10, SU-DHL-4, and SU-DHL-6). IL-21 stimulation induced tyrosine phosphorylation of STAT-1, -3, and -5 as early as fifteen minutes post treatment in DLBCL lines expressing either high (OCI-LY-3 and RCK-8) or low (SU-DHL-6 and OCI-LY-10) levels of IL-21R. In six of the seven DLBCL lines tested, IL-21 dramatically inhibited or completely abolished cellular proliferation at 25 ng/mL and 100 ng/mL concentrations, respectively. In contrast, one DLBCL cell line (OCI-LY-3) exhibited a threefold increase in cellular proliferation after stimulation with 25 ng/mL IL-21, but proliferation was effectively inhibited by 100 ng/mL IL-21. Marked apoptosis and cell death were observed by flow cytometry at 72 hours post IL-21 exposure in all nine NHL cell lines tested with the exception of OCI-LY-3 which exhibited no significant change in cell viability. The IL-21-induced apoptosis was associated with an activation of caspases 3/7, 8, and 9, detected as early as 12 hours post IL-21 exposure. IL-21 also induced an increase in the levels of the pro-apoptotic protein Bim in all the cell lines exhibiting marked apoptosis. In contrast, Bim protein levels decreased in response to the IL-21 stimulation in the resistant OCI-LY-3 DLBCL cell line. IL-21 treatment led to a decrease in the protein levels of Bcl-2 in all the analyzed cell lines except OCI-LY-3. An increase in expression of Bcl-6 protein in three of the four tested DLBCL cell lines was observed in response to IL-21 exposure. In primary tumors, IL-21 induced apoptosis in two of two DLBCLs, two of three follicular lymphomas, and two of six chronic lymphocytic leukemias. No apoptosis or cell death was induced in normal peripheral B-lymphocytes or in the HeLa or 293T control cells. These results suggest that IL-21 exhibits a direct anti-tumor effect on the DLBCL cell lines and primary tumors and point to a potential applicability of IL-21 in anti-DLBCL therapy. Further work interrogating the molecular mechanism of the IL-21-induced apoptosis of DLBCL cells in vitro and in animal models is in progress

HGAL, a Lymphoma Prognostic Biomarker, Regulates Lymphocyte and Lymphoma Cell Motility by Modulation of RhoA Signaling Pathway

Abstract Abstract 316 Identification of genes whose expression correlates with clinical outcome is of paramount importance for designing upfront patient-tailored treatments that will potentially improve the outcome of lymphoma patients. Furthermore, new molecular prognostic biomarkers may lead to the discovery of novel pathophysiological mechanisms underlying lymphoma pathogenesis, potentially highlighting unique biological processes regulated by these proteins in lymphoma cells and in their normal cellular counterparts. HGAL is a novel germinal center (GC)-specific gene whose expression predicts survival of patients with diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). Recently, we have demonstrated that HGAL is involved in negative regulation of lymphocyte migration, thus potentially constraining lymphocytes to the GC and preventing lymphoma dissemination; however, the molecular mechanism of HGAL effects on cell motility is unknown. Herein, we demonstrated that HGAL serves as a physiological regulator of the RhoA signaling pathway. HGAL over-expression or siRNA-mediated knock-down leads to increased or decreased levels of GTP-bound RhoA, respectively, but does not affect levels of GTP-bound CDC42 and RAC1. HGAL-induced activation of RhoA results in inhibition of lymphocyte and lymphoma cell motility and chemotaxis by activation of its downstream effector ROCK leading to: a) phosphorylation of myosin regulatory light chain (MRLC) that regulates actin-activated Mg-ATPase activity of myosin II; b) phosphorylation of myosin phosphatase (myosin PPTase) subunit MYPT1 inhibiting myosin light chain dephosphorylation; and c) phosphorylation of cofilin leading to cytoskeletal reorganization, as reflected by increased formation of focal adhesions and stress fibers. Opposite effects on RhoA downstream effectors are observed upon knock-down of HGAL expression. Furthermore, HGAL over-expression or knock-down modulate additional functions of RhoA, including transcriptional activation by serum response factor and RhoA transforming potential, as assessed by foci formation in NIH 3T3 cells. Co-immunoprecipitation experiments demonstrated that the effect of HGAL on RhoA is indirect and is mediated by RhoA-specific guanine nucleotide exchange factors (RhoGEFs) PDZ-RhoGEF and LARG that stimulate the GDP-GTP exchange rate. Moreover, we delineate the structural domains of HGAL and PDZ-RhoGEF that mediate the interaction between these proteins. Over-expression of HGAL mutants lacking the PDZ binding domain, mediating the interaction between HGAL and these RhoGEFs, fails to induce activation of RhoA and does not inhibit lymphoma cell motility and chemotaxis. Similarly, knock-downs of PDZ-RhoGEF and/or LARG reverse the inhibitory effects of HGAL on lymphoma cell motility and chemotaxis. These observations reveal a novel molecular mechanism underlying the inhibitory effects of HGAL on the motility of normal GC lymphocyte and GC-derived lymphoma cells. Since HGAL is specifically expressed only during the GC stage of B lymphocyte differentiation, these findings highlight the uniqueness and complexity of cell processes occurring during the GC reaction. They further elucidate the pathophysiologic mechanism underlying the favorable outcome of DLBCL and cHL patients whose tumors express high levels of HGAL protein. Disclosures: No relevant conflicts of interest to declare