Heterogeneous Pattern of Dependence on Anti-Apoptotic BCL-2 Family Proteins upon CHOP Treatment in Diffuse Large B-Cell Lymphoma

Expression of the anti-apoptotic B-cell lymphoma 2 (BCL-2) protein in patients with diffuse large B-cell lymphoma (DLBCL) strongly correlates with resistance to standard therapy with cyclophosphamide, vincristine, doxorubicin, prednisolone, and rituximab (R-CHOP). Although studies focus mainly on the contribution of BCL-2, here we also investigate the contribution of other anti-apoptotic proteins to CHOP-therapy resistance in DLBCL. Functional dynamic BCL-2 homology (BH)3 profiling was applied to DLBCL cell lines upon CHOP treatment or single CHOP compounds. Cell-specific anti-apoptotic dependencies were validated with corresponding BH3-mimetics. We found high expression of anti-apoptotic BCL-2, MCL-1, and BCL-XL in DLBCL cell lines and patients. CHOP treatment resulted in both enhanced and altered anti-apoptotic dependency. Enhanced sensitivity to different BH3-mimetics after CHOP treatment was confirmed in specific cell lines, indicating heterogeneity of CHOP-induced resistance in DLBCL. Analysis of single CHOP compounds demonstrated that similar changes could also be induced by doxorubicin or vincristine, providing evidence for clinical combination therapies of doxorubicin or vincristine with BH3-mimetics in DLBCL. In conclusion, we show for the first time that CHOP treatment induces increased anti-apoptotic dependency on MCL-1 and BCL-XL, and not just BCL-2. These results provide new perspectives for the treatment of CHOP-resistant DLBCL and underline the potential of BH3 profiling in predicting therapy outcomes

Targeting WEE1 in diffuse large B-cell lymphoma: mediator in DNA damage and apoptosis

Diffuse large B-cell lymphoma (DLBCL) is the most common and aggressive type of non-Hodgkin lymphoma. The current treatment for the disease currently cures only 60% of the patients, which means new therapies are needed to improve patient survival. In this project, we performed a large gene expression analysis in 1800 DLBCL patient samples to find new targets to improve the current treatment. Here we found high expression of the WEE1 protein, which is involved in cell cycle regulation and repair of DNA damage. Inhibition of the WEE1 protein in DLBCL cell lines induced disruption of the normal cell cycle and high levels of DNA damage, eventually causing cell death. In addition, WEE1 inhibition showed to enhance the currently used therapies for the treatment of DLBCL, which include rituximab (anti-CD20), radiation, CHOP (first line chemotherapy) and cytarabine (second line chemotherapy). Based on these findings, we also investigated the effect of WEE1 inhibition in combination with so-called “anti-apoptotic inhibitors”, which prevent cells from protecting themselves against cell death. These inhibitors are currently being tested in clinical trials for different types of non-Hodgkin lymphoma and leukemia’s, including DLBCL . We found that WEE1 inhibition worked very well together with the anti-apoptotic inhibitors, and that combination therapy significantly enhanced cell death in DLBCL. In total, our results demonstrate that inhibition of WEE1 is very successful in DLBCL, and would likely improve the current treatment available for DLBCL

The mitochondrial anti-apoptotic dependencies of hematologic malignancies: from disease biology to advances in precision medicine

Mitochondria are critical organelles in the regulation of intrinsic apoptosis. As a general feature of blood cancers, different anti-apoptotic members of the BCL-2 protein family localize at the outer mitochondrial membrane to sequester variable amounts of pro-apoptotic activators, and hence protect cancer cells from death induc-tion. However, the impact of distinct anti-apoptotic members on apop-tosis prevention, a concept termed anti-apoptotic dependence, differs remarkably across disease entities. Over the last two decades, several genetic and functional methodologies have been established to uncover the anti-apoptotic dependencies of the majority of blood cancers, inspiring the development of a new class of small molecules called BH3 mimetics. In this review, we highlight the rationale of targeting mito-chondrial apoptosis in hematology, and provide a comprehensive map of the anti-apoptotic dependencies that are currently guiding novel therapeutic strategies. Cell-extrinsic and -intrinsic mechanisms confer-ring resistance to BH3 mimetics are also examined, with insights on potential strategies to overcome them. Finally, we discuss how the field of mitochondrial apoptosis might be complemented with other dimen-sions of precision medicine for more successful treatment of ‘highly complex’ hematologic malignancies

Targeting tumor microenvironment crosstalk through GPCR receptors and PI3K pathway

[eng] The tumor microenvironment (TME) is gaining momentum due to its contribution to cancer progression and therapy resistance. This TME has a direct crosstalk with tumor cells that involves the activation of different pathways. Follicular lymphoma (FL) is the most common indolent non-Hodgkin lymphoma. Although FL is generally characterized by slow progression and high response rates to therapy, it is still considered incurable, because almost virtually all the patients relapse. FL is probably the NHL with the highest dependence on microenvironment. PI3K is a common denominator transducing the signaling from FL crosstalk with the TME and plays an important role in multiple cellular functions, and also contributes to cancer promoting aspects of the TME, such as angiogenesis and inflammatory cell recruitment. Idelalisib is a first-in-class δ isoform- specific PI3K inhibitor that receive regulatory approval for relapsed CLL, SLL and FL in 2014. Idelalisib blocks PI3K δ which is restricted to leukocytes. BCL2 deregulation is paramount in the pathogenesis of FL, as a consequence of the t(14;18), and therefore it is an attractive target for novel therapeutic approaches. Venetoclax (ABT-199, AbbVie) is a small BCL-2 inhibitors. Even though 85% of FL patients harbor the t(14;18), the results of the first clinical trial with venetoclax were not satisfactory (overall response 38%). From this first study we conclude that Idelalisib modulates key pathways in the germinal center and shapes the FL immune microenvironment by decreasing the recruitment of TFH and Treg to the tumor site leading to less immunesuppresive phenotype. Furthermore Idelalisib induces a moderate cytotoxic effect on FL cells in co-cultures. This co-culture decrease FL dependence on BCL-2 and consequently, venetoclax cytotoxicity, but Idelalisib sensitizes FL co-cultures to venetoclax. In summary, Idelalisib interferes with the crosstalk of FL and its immune microenvironment and potentiates the activity of venetoclax targeting the tumor cells, thus representing a promising combination therapy that may improve FL outcome. Colorectal cancer (CRC) is the third most common cancer in males and the second in females, and the fourth most common cause of cancer-related death worldwide. Patients with advanced and distal metastatic disease (stage IV), the survival rate drops to 10%, which accounts for approximately 18% of cases. The TME in CRC, is a complex structure composed by different type of cells, which are interacting each other’s and secreting a variety of growth factors and other molecules, such as cytokines and chemokines. Tumor development is based on the crosstalk between tumor cells and their surrounding microenvironment, and this crosstalk is mediated by the receptors and its ligand expression in both types of cells. G protein-coupled receptors (GPCRs) are an important family of membrane signaling receptors, which have an important role in cancer growth and development. Originally, GPCRs were considered as monomeric functional entities, nevertheless, in recent years has become evident that GPCRs form dimers and this dimers formation may modify the cellular response. In cancer, CXCR4 (has been studied extensively) plays an important role at different stages of cancer development, and is involved in the metastasis process of tumor cells. The up- regulation of CXCR4 in CRC correlates with a poor prognosis. Another GPCR, CB2 receptor modulates the downstream signaling and it is able to activate a wide range of signaling pathways, including extracellular signal-regulated kinases 1/2 (ERK1/2). In CRC, it has been described an up-regulation of CB2 receptor expression. GPCRs show differential expression in cancer cells and tissues, and they are highly druggable sites. From this second study we concluded that CXCR4 and CB2 expression is increased in primary colon tumor cells and in metastasis cells compared to normal epithelial cells from colon mucosa, and they formed heterodimers in colon tumoral cells and are associated with more aggressive phenotypes. Moreover, a bidirectional cross-antagonism crosstalk is established between these receptors. These heterodimers regulate in vitro CXCL12-induced migration, and in vivo, the simultaneous inhibition of both receptors shows superior anti-tumoral and anti-metastatic activities than the single agent inhibition. In summary, targeting the heterodimerization of CXCR4 and CB2 that are biologically relevant in cancer can be an effective way to reduce proliferation and dissemination in CRC.[spa] El estudio del microambiente tumoral está ganando importancia en las últimas décadas debido a su contribución en la formación y desarrollo del cáncer, además de contribuir en la resistencia de las células tumorales a diferentes terapias. Este microambiente interactúa con las células tumorales y activa diferentes vías. El linfoma folicular (FL), es el linfoma no Hodgkin indolente más común y con mayor dependencia del microambiente tumoral, además es considerado incurable. PI3K desempeña un papel importante en la comunicación con el microambiente, y es importante en múltiples funciones celulares, además de contribuir en la angiogénesis, reclutamiento de células inflamatorias y promover el crecimiento tumoral. Idelalisib es un inhibidor de PI3K (específicamente de la isoforma δ), que se aprobó en 2014 por la FDA. Paralelamente la desregulación de BCL2 es primordial en la patogénesis de FL, como consecuencia de la t (14; 18), presente en un 85% de los pacientes, y por lo tanto es un objetivo atractivo para novedosos enfoques terapéuticos. Venetoclax (ABT-199, AbbVie) es un pequeño inhibidor de BCL2, que mostró unos resultados del primer ensayo clínico no satisfactorios (respuesta global del 38%). De este primer estudio concluimos que Idelalisib interfiere en la comunicación de FL y su microambiente inmune, además potencia la actividad de venetoclax atacando a las células tumorales, lo que representa una terapia de combinación prometedora que puede mejorar el resultado del tratamiento de FL

Elucidation of the Role of DNA-Damage Response Genes in the Tumor Microenvironment and Molecular Characterization of a “Tropical” Chronic Lymphocytic Leukemia Cohort

In most human cancers such as diffuse large B cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), major genes of the DNA damage response (DDR) complex such as TP53, suffers inactivating mutations and dysregulation that impairs DNA repair processes and apoptotic machinery relevant for treatment. Despite the successes with Chemoimmunotherapy (CIT), many B-cells malignancies remain a major clinical challenge as therapy resistance account for 90% of cancer related fatalities. The tumor microenvironment (TME) is particularly culpable. Protumorigenic TME promotes complex crosstalk of tumor and stroma cells to facilitate tumor survival, proliferation, immune escape and metastasis. This study elucidated the role of DDR in TME interactions and characterized molecular signature of Ghanaian CLL cohort. Using shRNA to downregulate DDR genes in an ABC-subtype of DLBCL cell line we demonstrate that, functional TP53 and ATX (SMG-1) genes are critical for ADCP of DLBCL cells and that loss of TP53 and ATX in DLBCL induce resistance towards CIT by inhibition of macrophage effector functions through changes in formation/secretion of cellular secretome. Additionally, phagocytosis is significantly enhanced in ABC-DLBCL expressing high levels of PD-L1 and CD47 antigens when treated in vitro with CIT/anti-CD47, anti-PD-L1 or anti-PD1 combination. Particularly, CD47/Sirp-α blockade circumvent impaired ADCP due to TP53, ATX, KD providing a rational for incorporation of checkpoint inhibitors into current R-CHOP regimen for the management of TP53, ATX mutated, resistant/refractory ABC-DLBCL. Characterization of tropical African CLL showed females predominant incidence (51.1% vs 48.9%), lower median age of incidence (59yrs with 42.2% < 55yrs) compared to Western CLL which shows predominant males incidence (2:1) with a higher median age (71yrs, with 5-11% < 55yrs). Measurement of informative molecular markers showed 80% good prognosis by ZAP-70 and CD38 but 88.4% presentation at late clinical stage of disease, higher monotypic sIgM than dual IgM+/IgD+ (54.5% vs 34.1%) compared to predominant dual IgM+/IgD+ expression among European patients. These differences in CLL biology and clinical outcome may reflect interplay of genetic and environmental factors among racial groups in different geographical settings

Targeting Oncogenic Protein Sythesis in MYC-driven B Cell Lymphoma

Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) are aggressive tumors of mature B cells that can be distinguished based on histomorphological, phenotypic, and genetic features. B-cell lymphoma unclassifiable, with features intermediate between BL and DLBCL (BCL-U) is a subset of mature B cell lymphoma contains one or more features that overlap BL and DLBCL. Previous molecular analyses prove there is a biological continuum between BL and DLBCL based on the activity of MYC. Herein, in order to resolve the molecular heterogeneity of BCL-U, we tested whether a targeted expression profiling panel could categorize tumors as BL and DLBCL. Among these MYC driven lymphomas, those with concurrent MYC and BCL2 dysregulation are especially a challenge in clinical practice due to rapid disease progression, resistance to standard chemotherapy and high risk of refractory. MYC plays a central role by coordinating hyperactive protein synthesis with upregulated transcription in order to support the rapid proliferation. Translation initiation inhibitor rocaglates were identified as the most potent drug across all MYC driven lymphomas as they efficiently inhibit MYC expression and tumor cell viability. Surprisingly, we found this class of compounds does not inhibit translation by depleting the abundance of eIF4A in eIF4F, but more likely acts as a selective roadblock to prevent ribosome scanning. Furthermore, proteome wide quantification demonstrated rocaglate induced selective repression of multiple critical oncoproteins beside MYC in B cell lymphoma including NEK2, MCL1, AURKA, PLK1 and several transcription factors that generally considered undruggable. Finally, (-)-SDS-1-021, the most promising rocaglate, was confirmed highly potent as single agent, and displayed significant synergy with BCL2 inhibitor ABT199 in inhibiting tumor growth and survival in patient derived xenograft mouse models. Overall, our findings support the strategy of using rocaglates to target oncoprotein synthesis in MYC-driven lymphomas

Discovery of novel molecular and biochemical predictors of response and outcome in diffuse large B-cell lymphoma

PhDDiscovery of Novel Molecular and Biochemical Predictors of Response and Outcome in Diffuse Large B-cell Lymphoma Diffuse large B-cell lymphoma (DLBCL) is the commonest form of non-Hodgkin lymphoma and responds to treatment with a 5-year overall survival (OS) of 40-50%. Predicting outcome using the best available method, the International Prognostic Index (IPI), is inaccurate and unsatisfactory. This thesis describes research undertaken to discover, explore and validate new molecular and biochemical predictors of response and long-term outcome with the aims of improving on the inaccurate IPI and of suggesting novel therapeutic approaches. Two strategies were adopted: a rational and an empirical approach. The rational strategy used gene expression profiling to identify transcriptional signatures that correlated with outcome to treatment and from which a model of 13-genes accurately predict long-term OS. Two components of the 13-gene model, PKC and PDE4B, were studied using inhibitors in lymphoma cell-lines and primary cell cultures. PKC inhibition using SC-236 proved to be cytostatic and cytotoxic in the cell-lines examined and to a lesser extent in primary tumours. PDE4 inhibition using piclamilast and rolipram had no effect either alone or in combination with chemotherapy. The empirical approach investigated the trace element selenium in presentation serum and found that it was a biochemical predictor of response and outcome to treatment. In an attempt to provide evidence of a causal relationship as an explanation for the associations between presentation serum selenium, response and outcome, two selenium compounds, methylseleninic acid (MSA) and selenodiglutathione (SDG) were studied in vitro in the same lymphoma cell-lines and primary cell cultures. Both MSA and SDG exhibited cytostatic and cytotoxic activity and caspase-8 and caspase-9 driven apoptosis. For SDG reactive oxygen species generation was important for its activity in three of the four cell-lines. In conclusion, molecular and biochemical predictors of response and survival were discovered in DLBCL that led to viable targets for drug intervention being validated in vitro

NF-kappaB-dependent regulation of the diagnostic marker CD10 and role of BCL-2 activity in histone deacetylase inhibitor-induced apoptosis in human B-lymphoma cell lines

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease with multiple distinct molecular subtypes. Increased NF-κB activity and expression of the microRNA miR-155 (product of the BIC gene) are associated with one subtype, called the activated B-cell (ABC) subtype. It is shown here that induction of NF-κB activity leads to increased miR-155 expression, the levels of miR-155 in a panel of B-lymphoma cell lines correlate with increased NF-κB activity, and the NF-κB p50/p65 heterodimer binds to a specific DNA site in the BIC promoter. Also described is a regulatory network wherein NF-κB-dependent up-regulation of miR-155 leads to reduced PU.1 transcription factor expression and consequently reduced PU.1-driven expression of B-lymphoma marker CD10 in the human B-lymphoma cell line BJAB. Genetic variation in DLBCL can be used to explain the response of individual patients to chemotherapy. One cancer therapeutic approach currently in clinical trials uses histone deacetylase inhibitors (HDACi's) as a monotherapy or in combination with other vi agents. It is shown here that two pan-HDACi's, trichostatin A and vorinostat, induce apoptosis in seven of eight human DLBCL cell lines. Ectopic over-expression of antiapoptotic proteins BCL-2 and BCL-XL or the pro-apoptotic protein BIM in select DLBCL cell lines can confer further resistance or sensitivity, respectively, to HDACi treatment. Additionally, the BCL-2 family antagonist ABT-737 can increase the sensitivity of several DLBCL cell lines to vorinostat-induced apoptosis, including the HDACi-resistant SUDHL6 cell line. Moreover, one vorinostat-resistant variant of the HDACi-sensitive cell line SUDHL4 has increased expression of anti-apoptotic proteins BCL-XL and MCL-1 and decreased sensitivity to ABT-737, and a second such variant cell line has increased expression of anti-apoptotic protein MCL-1. These results suggest that the balance of anti- to pro-apoptotic BCL-2 family protein expression is important in determining the sensitivity of DLBCL cell lines to HDACi-induced apoptosis. Thus, the sensitivity of DLBCL cell lines to treatment with HDACi's appears to depend on the complex regulation of BCL-2 family members, suggesting that the response of a subset of DLBCL patients to HDACi treatment may benefit from co-treatment with BCL-2 antagonists

An Autochthonous Mouse Model of Myd88- and BCL2-Driven Diffuse Large B-cell Lymphoma Reveals Actionable Molecular Vulnerabilities

Diffuse large B-cell lymphoma is the most common non-Hodgkin`s lymphoma and accounts for 30-40% of newly diagnosed lymphoma. DLBCL can be subdivided in the activated B-cell-like (ABC) and the germinal center B-cell-like (GCB) DLBCL. Large-scale sequencing efforts showed a large overlap between ABC-and GCB-DLBCL. Therefore two research groups recently published new genetic based approaches to classify DLBCL into distinct clusters. The most aggressive cluster in regards to clinical outcome and resistance to first line treatment is the MCD/C5-DLBCL, enriched with former ascribed ABC-DLBCL cases. Two of the most common aberrations in MCD/C5-DLBCL are a copy-number gain of BCL2and a point mutation in MYD88.In this study we present a detailed characterization of our Myd88-and BCL2-driven MCD/C5-DLBCL mouse model. We characterized the mouse model using immune phenotyping, RNA sequencing and whole exome sequencing. We could show that the lymphomas derived from our mouse model resemble genetic features of human MCD/C5-DLBCL. We detected that Myd88and BCL2induce splenomegaly and germinal center formation in vivo. This indicates that the oncogenic Myd88and BCL2overexpression might cooperate in lymphomagenesis. Furthermore we could detect an actionable dependenceof the murine MCD/C5-DLBCL model on BCL2. This BCL2dependence was also shown in human MCD/C5-DLBCL cell lines. Moreover, the human MCD/C5-DLBCL in comparison to the human non-MCD/C5-DLBCL showed a higher expression of PD-L1. Therefore we used our MCD/C5-DLBCL mouse model as preclinical tool to test a combined blockade of BCL2 and PD-1. We observed a significant synergistic effect of the combined treatment in comparison to the single agents and the vehicle control with regard to overall survival and tumor growth control, which could be shown by MRI monitoring.Altogether, we demonstrated that our Myd88/BCL2-driven mouse model resembles many features of the human MCD/C5-DLBCL. Further we could use our MCD/C5-DLBCL mouse model as a preclinical tool to detect actionable molecular vulnerabilities and to test new treatment regimens. Based on the detected cooperation of mutant Myd88and BCL2in murine lymphomagenesis, we were able to show a synergistic effect of combined BCL2 and PD-1 blockade in murine MCD/C5-DLBCL. These data indicate that it could be a reasonable approach to test a combined BCL2/PD-1 blockade inrelapsed or refractory patients