Innovative targeted therapies for chemorefractory B-cell non-Hodgkin lymphomas

[eng] Lymphomas are a heterogeneous group of tumors characterized by the proliferation of lymphocytes predominantly in lymphoid structures but also in extranodal tissues. More than 90% of patients are afflicted by lymphomas of B-cell origin. The current World Health Organization (WHO) classification of hematopoietic and lymphoid tumors categorizes B-cell neoplasms in more than 40 distinct disease entities, according to a combination of the morphology, immunophenotype, genetic, molecular and clinical features. Each entity has its own clinical course and requires specific treatments. The characterization of activated signaling pathways involved in survival and proliferation, together with the development of a wide pharmacological armamentarium against cancer, have facilitated the bench-to-bedside translation of new targeted therapies in B-cell non-Hodgkin lymphomas (B-NHL). These novel therapies include two of the most relevant drugs lately approved: the anti-apoptotic agent venetoclax and the BTK inhibitor ibrutinib. One major hurdle to their successful application is the rise of drug resistance. Resistance to therapy is observed in many cases of B-cell malignancies. This phenomenon significantly limits the utility of the current therapeutic strategies, and remains a substantial challenge for the clinical management of patients with advanced cancers. Resistance comes in two flavors: intrinsic resistance (also known as innate or de novo resistance) and acquired resistance, resulting from the clonal evolution of resistant variants. With this concept in mind, in this thesis we have explored new approaches to overcome the development of drug resistance. Venetoclax (ABT-199) is a first-in-class BH3 mimetic, FDA-approved for use in patients with R/R del17p chronic lymphocytic leukemia. In the clinical setting, it has demonstrated high response rates and good toxicity profiles in other subtypes of relapsed/refractory non-Hodgkin lymphoma. We proposed a model of double hit lymphoma (DHL) resistance to ABT-199 in which the capacity of CPI203 to regulate the transcriptome of the cells could help to circumvent this problem. In ABT-199 sensitive cells, the BH3 mimetic acts by displacing BIM from BCL-2 complexes, allowing the de-repression and/or direct activation of BAX and leading to an activation of mitochondrial outer membrane permeabilization. In DHL cells, a compensatory upregulation of BFL-1 would bind and inactivate the pool of BIM proteins released from BCL-2 by ABT-199, avoiding MOMP and preserving cell survival. CPI203 primes cells to death by decreasing BFL-1 and increasing BIM protein levels, and its combination with ABT-199 allows to tip the balance between pro- and anti-apoptotic signaling toward induction of cell death. This concept provides a new insight in the proposed mechanisms of resistance to BH3- mimetics in NHL cell lines, where MCL-1 and BCL-XL have been proposed as the major determinants of drug sensitivity and acquired resistance. On the other hand, ibrutinib is a BTK inhibitor approved for first-line therapy in patients with chronic lymphocytic leukemia, as well as for the treatment of some relapsed/refractory B-NHL. Despite its high level of clinical activity, acquiring of mutations or re-wiring of the BCR pathway to retain the downstream signaling appears to be a common mechanism of resistance. The inhibition of more than one BCR kinase might be useful in B-NHL cases that are resistant to the sole inhibition of BTK. We describe the compound IQS019 as a new BCR kinase inhibitor able to counteract both constitutive and ligand-dependent activation of the BCR pathway. Its capacity to inhibit the three upstream BCR kinases, BTK, SYK and LYN, confer an advantage over the inhibition of BTK alone by ibrutinib, in in vitro and in vivo models of B-NHL, being of special importance for the treatment of those patients with non-canonical NF-κB activation, who are low responders to ibrutinib. Therefore, the development of innovative therapeutic approaches that permit to overcome drug resistance opens a window to important therapeutic advances in the treatment of B-NHL.[spa] Las neoplasias linfoides de célula B constituyen un grupo heterogéneo de tumores caracterizados por la proliferación de linfocitos B. Cada entidad clínica posee unas características particulares y requiere de un tratamiento específico. A pesar de los importantes avances terapéuticos, la supervivencia a largo plazo sigue siendo baja y precisa de un desarrollo constante de nuevas aproximaciones terapéuticas. Uno de los mayores problemas asociados a la respuesta a fármacos son las resistencias. En muchos casos estas resistencias se deben a cambios en proteínas diana o a la modulación compensatoria de otras proteínas o vías de señalización. El conocimiento de estos cambios será de gran importancia para poder encontrar aproximaciones terapéuticas que permitan eliminar estas resistencias. El linfoma doble-hit es un linfoma agresivo caracterizado por su baja respuesta a la quimioterapia estándar. Entre los múltiples agentes terapéuticos específicos actualmente en desarrollo encontramos el inhibidor de BCL-2, venetoclax. El venetoclax ha demostrado ser efectivo en varios subtipos de linfoma pero su uso conlleva el problema de la aparición de resistencias. Varios estudios han destacado el papel de proteínas de la familia BCL-2 en este proceso. Nuestros resultados indican que la regulación positiva de BFL-1 es uno de los factores clave en el desarrollo de resistencias al fármaco. Su regulación mediante el CPI203, un modulador epigenético, resulta en una sensibilización al venetoclax, tanto in vitro como in vivo. Por otro lado, la señalización de los receptores de células B (BCR) contribuye a la patogénesis de las neoplasias malignas de células B y ha surgido como una nueva diana terapéutica en varios tipos de linfoma. Así, los inhibidores de quinasas de la vía del BCR constituyen una estrategia terapéutica prometedora. Dentro de este grupo de fármacos destaca el inhibidor de Btk ibrutinib, que ha conseguido esperanzadoras tasas de respuesta pero que también se ve afectado por la aparición de resistencias. Nuestro trabajo muestra que el compuesto IQS019, inhibidor de varias quinasas de la vía del BCR (Btk, Syk y Lyn), posee un potente efecto antitumoral y permite escapar a las resistencias observadas al ibrutinib. Así, supone un buen tratamiento para varios subtipos de linfomas de células B, incluyendo aquellos poco sensibles a los inhibidores de quinasa de BCR actuales

A mouse model of disseminated mantle cell lymphoma highlights a lack of activity of estrogen receptor β agonists toward tumor burden

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ZEB1-induced tumourigenesis requires senescence inhibition via activation of DKK1/mutant p53/Mdm2/CtBP and repression of macroH2A1.

OBJECTIVE: Understand the role of ZEB1 in the tumour initiation and progression beyond inducing an epithelial-to-mesenchymal transition. DESIGN: Expression of the transcription factor ZEB1 associates with a worse prognosis in most cancers, including colorectal carcinomas (CRCs). The study uses survival analysis, in vivo mouse transgenic and xenograft models, gene expression arrays, immunostaining and gene and protein regulation assays. RESULTS: The poorer survival determined by ZEB1 in CRCs depended on simultaneous high levels of the Wnt antagonist DKK1, whose expression was transcriptionally activated by ZEB1. In cancer cells with mutant TP53, ZEB1 blocked the formation of senescence-associated heterochromatin foci at the onset of senescence by triggering a new regulatory cascade that involves the subsequent activation of DKK1, mutant p53, Mdm2 and CtBP to ultimately repress macroH2A1 (H2AFY). In a transgenic mouse model of colon cancer, partial downregulation of Zeb1 was sufficient to induce H2afy and to trigger in vivo tumour senescence, thus resulting in reduced tumour load and improved survival. The capacity of ZEB1 to induce tumourigenesis in a xenograft mouse model requires the repression of H2AFY by ZEB1. Lastly, the worst survival effect of ZEB1 in patients with CRC ultimately depends on low expression of H2AFY and of senescence-associated genes. CONCLUSIONS: The tumourigenic capacity of ZEB1 depends on its inhibition of cancer cell senescence through the activation of a herein identified new molecular pathway. These results set ZEB1 as a potential target in therapeutic strategies aimed at inducing senescence

Activity of the novel BCR kinase inhibitor IQS019 in preclinical models of B-cell non-Hodgkin lymphoma

Background Pharmacological inhibition of B cell receptor (BCR) signaling has recently emerged as an effective approach in a wide range of B lymphoid neoplasms. However, despite promising clinical activity of the first Bruton’s kinase (Btk) and spleen tyrosine kinase (Syk) inhibitors, a small fraction of patients tend to develop progressive disease after initial response to these agents. Methods We evaluated the antitumor activity of IQS019, a new BCR kinase inhibitor with increased affinity for Btk, Syk, and Lck/Yes novel tyrosine kinase (Lyn), in a set of 34 B lymphoid cell lines and primary cultures, including samples with acquired resistance to the first-in-class Btk inhibitor ibrutinib. Safety and efficacy of the compound were then evaluated in two xenograft mouse models of B cell lymphoma. Results IQS019 simultaneously engaged a rapid and dose-dependent de-phosphorylation of both constitutive and IgM-activated Syk, Lyn, and Btk, leading to impaired cell proliferation, reduced CXCL12-dependent cell migration, and induction of caspase-dependent apoptosis. Accordingly, B cell lymphoma-bearing mice receiving IQS019 presented a reduced tumor outgrowth characterized by a decreased mitotic index and a lower infiltration of malignant cells in the spleen, in tight correlation with downregulation of phospho-Syk, phospho-Lyn, and phospho-Btk. More interestingly, IQS019 showed improved efficacy in vitro and in vivo when compared to the first-in-class Btk inhibitor ibrutinib, and was active in cells with acquired resistance to this latest. Conclusions These results define IQS019 as a potential drug candidate for a variety of B lymphoid neoplasms, including cases with acquired resistance to current BCR-targeting therapies

Cytoplasmic cyclin D1 controls the migration and invasiveness of mantle lymphoma cells

Abstract Mantle cell lymphoma (MCL) is a hematologic neoplasm characterised by the t(11;14)(q13;q32) translocation leading to aberrant cyclin D1 expression. The cell functions of cyclin D1 depend on its partners and/or subcellular distribution, resulting in different oncogenic properties. We observed the accumulation of cyclin D1 in the cytoplasm of a subset of MCL cell lines and primary cells. In primary cells, this cytoplasmic distribution was correlated with a more frequent blastoid phenotype. We performed immunoprecipitation assays and mass spectrometry on enriched cytosolic fractions from two cell lines. The cyclin D1 interactome was found to include several factors involved in adhesion, migration and invasion. We found that the accumulation of cyclin D1 in the cytoplasm was associated with higher levels of migration and invasiveness. We also showed that MCL cells with high cytoplasmic levels of cyclin D1 engrafted more rapidly into the bone marrow, spleen, and brain in immunodeficient mice. Both migration and invasion processes, both in vivo and in vitro, were counteracted by the exportin 1 inhibitor KPT-330, which retains cyclin D1 in the nucleus. Our data reveal a role of cytoplasmic cyclin D1 in the control of MCL cell migration and invasion, and as a true operator of MCL pathogenesis

Additional file 1: of Activity of the novel BCR kinase inhibitor IQS019 in preclinical models of B-cell non-Hodgkin lymphoma

Figure S1. IQS019 tyrosine kinase inhibitory profiling. Tyrosine kinase (TK) and tyrosine kinase-like (TKL) kinome tree was elaborated on the basis of residual in vitro kinase activity upon exposure to 100 nM or 1 μM IQS019, by means of Kinome Render software ( http://bcb.med.usherbrooke.ca/kinomerender.php ). Figure S2. Sensitivity of CLL primary cases to IQS019 is independent of IGHV mutational status and involves a caspase-dependent cell death process. (a) CLL primary cells, 9 of them with ummutated (UM) and 6 with mutated (M) IGHV gene, were treated with increasing concentrations of IQS019 for 24h. Cell viability was determined by MTT method. Shown are the median values from each CLL group (UM and M), referred to control, untreated cells. (b) IQS019 induces caspase-dependent cell death in MCL (UPN-1) and in FL (DOHH-2) cell lines, as well as in two representative CLL primary cultures. Cells were exposed for 24 hours to 5 μM IQS019, in the presence of absence of the pan-caspase inhibitor Q-VD-OPh (10 μM). Apoptosis was determined by simultaneous cytofluorimetric detection of Annexin-V and caspase-3/7 activity. (c) A set of 6 CLL primary cultures were treated with IQS019 as indicated, followed by Western Blot detection of phospho-histone H3 (p-H3), using β- actin as a loading control. Figure S3. Flow cytometry determination of CXCR4 membrane expression in B-NHL cell lines. Four representative cell lines were stained with a PE-labeled anti-CXCR4 antibody and analyzed on an Attune cytometer. CXCR4-specific signal (black curves) and isotypic control (grey filled curve) are represented. Figure S4. Safety and PK properties of IQS019-2MeSO3H in mice. (a) Twenty SCID mice (10 males and 10 females) received a single intravenous injection of IQS019-2MeSO3H at a 2 mg/kg, 10 mg/kg, or 50 mg/kg dose, or equivalent volume of vehicle, and animal weight was recorded at days 1, 3, 4, 7, 11, 14, 18 and 21 post-treatment. (b) Mean plasma concentration of IQS019-2MeSO3H in ICR mice over the time, after a single p.o. administration of a 25 mg/kg dose of the compound. Figure S5. Comparison of parental and ibrutinib-resistant derived B-NHL cell line. (a) Dose-response of the UPN-1 parental, and UPN-IbruR derived cell line exposed for 72 hours to increasing concentrations of ibrutinib or IQS019. (b) BTK and PLCG2 exon sequencing in UPN-IbruR cells. (c) Western blot detection of the alternative NF-κB pathway component, p52, in UPN-1 and UPN-IbruR cells. β-actin was used as a loading control. (DOC 3279 kb

Pharmacological modulation of CXCR4 cooperates with BET bromodomain inhibition in diffuse large B-cell lymphoma

Constitutive activation of the chemokine receptor CXCR4 has been associated with tumor progression, invasion, and chemotherapy resistance in different cancer subtypes. Although the CXCR4 pathway has recently been suggested as an adverse prognostic marker in diffuse large B-cell lymphoma, its biological relevance in this disease remains underexplored. In a homogeneous set of 52 biopsies from patients, an antibody-based cytokine array showed that tissue levels of CXCL12 correlated with high microvessel density and bone marrow involvement at diagnosis, supporting a role for the CXCL12-CXCR4 axis in disease progression. We then identified the tetra-amine IQS-01.01RS as a potent inverse agonist of the receptor, preventing CXCL12-mediated chemotaxis and triggering apoptosis in a panel of 18 cell lines and primary cultures, with superior mobilizing properties in vivo than those of the standard agent. IQS-01.01RS activity was associated with downregulation of p-AKT, p-ERK1/2 and destabilization of MYC, allowing a synergistic interaction with the bromodomain and extra-terminal domain inhibitor, CPI203. In a xenotransplant model of diffuse large B-cell lymphoma, the combination of IQS-01.01RS and CPI203 decreased tumor burden through MYC and p-AKT downregulation, and enhanced the induction of apoptosis. Thus, our results point out an emerging role of CXCL12-CXCR4 in the pathogenesis of diffuse large B-cell lymphoma and support the simultaneous targeting of CXCR4 and bromodomain proteins as a promising, rationale-based strategy for the treatment of this disease