Teresa Poggio

Phosphatidylinositol 3-kinase δ blockade increases genomic instability in B cells

Activation-induced cytidine deaminase (AID) is a B-cell-specific enzyme that targets immunoglobulin genes to initiate class switch recombination and somatic hypermutation. In addition, through off-target activity, AID has a much broader effect on genomic instability by initiating oncogenic chromosomal translocations and mutations involved in the development and progression of lymphoma. AID expression is tightly regulated in B cells and its overexpression leads to enhanced genomic instability and lymphoma formation. The phosphatidylinositol 3-kinase δ (PI3Kδ) pathway regulates AID by suppressing its expression in B cells. Drugs for leukaemia or lymphoma therapy such as idelalisib, duvelisib and ibrutinib block PI3Kδ activity directly or indirectly, potentially affecting AID expression and, consequently, genomic stability in B cells. Here we show that treatment of primary mouse B cells with idelalisib or duvelisib, and to a lesser extent ibrutinib, enhanced the expression of AID and increased somatic hypermutation and chromosomal translocation frequency to the Igh locus and to several AID off-target sites. Both of these effects were completely abrogated in AID-deficient B cells. PI3Kδ inhibitors or ibrutinib increased the formation of AID-dependent tumours in pristane-treated mice. Consistently, PI3Kδ inhibitors enhanced AID expression and translocation frequency to IGH and AID off-target sites in human chronic lymphocytic leukaemia and mantle cell lymphoma cell lines, and patients treated with idelalisib, but not ibrutinib, showed increased somatic hypermutation in AID off-targets. In summary, we show that PI3Kδ or Bruton’s tyrosine kinase inhibitors increase genomic instability in normal and neoplastic B cells by an AID-dependent mechanism. This effect should be carefully considered, as such inhibitors can be administered to patients for years.

ALK-Dependent Control of Hypoxia-Inducible Factors Mediates Tumor Growth and Metastasis

Rearrangements involving the anaplastic lymphoma kinase (ALK) gene are defining events in several tumors, including anaplastic large-cell lymphoma (ALCL) and non-small cell lung carcinoma (NSCLC). In such cancers, the oncogenic activity of ALK stimulates signaling pathways that induce cell transformation and promote tumor growth. In search for common pathways activated by oncogenic ALK across different tumors types, we found that hypoxia pathways were significantly enriched in ALK-rearranged ALCL and NSCLC, as compared with other types of T-cell lymphoma or EGFR- and K-RAS-mutated NSCLC, respectively. Consistently, in both ALCL and NSCLC, we found that under hypoxic conditions, ALK directly regulated the abundance of hypoxia-inducible factors (HIF), which are key players of the hypoxia response in normal tissues and cancers. In ALCL, the upregulation of HIF1α and HIF2α in hypoxic conditions required ALK activity and its downstream signaling proteins STAT3 and C/EBPβ. In vivo, ALK regulated VEGFA production and tumor angiogenesis in ALCL and NSCLC, and the treatment with the anti-VEGFA antibody bevacizumab strongly impaired ALCL growth in mouse xenografts. Finally, HIF2α, but not HIF1α, was required for ALCL growth in vivo whereas the growth and metastasis potential of ALK-rearranged NSCLC required both HIF1α and HIF2α. In conclusion, we uncovered an ALK-specific regulation of the hypoxia response across different ALK(+) tumor types and propose HIFs as a powerful specific therapeutic target in ALK-rearranged ALCL and NSCLC.

Modeling Lung Cancer Evolution and Preclinical Response by Orthotopic Mouse Allografts

Cancer evolution is a process that is still poorly understood because of the lack of versatile in vivo longitudinal studies. By generating murine non-small cell lung cancer (NSCLC) orthoallobanks and paired primary cell lines, we provide a detailed description of an in vivo, time-dependent cancer malignization process. We identify the acquisition of metastatic dissemination potential, the selection of co-driver mutations, and the appearance of naturally occurring intratumor heterogeneity, thus recapitulating the stochastic nature of human cancer development. This approach combines the robustness of genetically engineered cancer models with the flexibility of allograft methodology. We have applied this tool for the preclinical evaluation of therapeutic approaches. This system can be implemented to improve the design of future treatments for patients with NSCLC.

Efficacy of a Cancer Vaccine against ALK-Rearranged Lung Tumors

Lung cancers harboring ALK translocations are treated with protein kinase inhibitors, which can extend survival. A cancer vaccine against ALK induced strong immune responses and enhanced survival when used alone, or in combination with kinase inhibitors or checkpoint inhibitors. Non–small cell lung cancer (NSCLC) harboring chromosomal rearrangements of the anaplastic lymphoma kinase (ALK) gene is treated with ALK tyrosine kinase inhibitors (TKI), but the treatment is successful for only a limited amount of time; most patients experience a relapse due to the development of drug resistance. Here, we show that a vaccine against ALK induced a strong and specific immune response that both prophylactically and therapeutically impaired the growth of ALK-positive lung tumors in mouse models. The ALK vaccine was efficacious also in combination with ALK TKI treatment and significantly delayed tumor relapses after TKI suspension. We found that lung tumors containing ALK rearrangements induced an immunosuppressive microenvironment, regulating the expression of PD-L1 on the surface of lung tumor cells. High PD-L1 expression reduced ALK vaccine efficacy, which could be restored by administration of anti–PD-1 immunotherapy. Thus, combinations of ALK vaccine with TKIs and immune checkpoint blockade therapies might represent a powerful strategy for the treatment of ALK-driven NSCLC. Cancer Immunol Res; 3(12); 1333–43. ©2015 AACR.

Excess of NPM-ALK oncogenic signaling promotes cellular apoptosis and drug dependency

Most of the anaplastic large-cell lymphoma (ALCL) cases carry the t(2;5; p23;q35) that produces the fusion protein NPM-ALK (nucleophosmin-anaplastic lymphoma kinase). NPM-ALK-deregulated kinase activity drives several pathways that support malignant transformation of lymphoma cells. We found that in ALK-rearranged ALCL cell lines, NPM-ALK was distributed in equal amounts between the cytoplasm and the nucleus. Only the cytoplasmic portion was catalytically active in both cell lines and primary ALCL, whereas the nuclear portion was inactive because of heterodimerization with NPM1. Thus, about 50% of the NPM-ALK is not active and sequestered as NPM-ALK/NPM1 heterodimers in the nucleus. Overexpression or relocalization of NPM-ALK to the cytoplasm by NPM genetic knockout or knockdown caused ERK1/2 (extracellular signal-regulated protein kinases 1 and 2) increased phosphorylation and cell death through the engagement of an ATM/Chk2- and γH2AX (phosphorylated H2A histone family member X)-mediated DNA-damage response. Remarkably, human NPM-ALK-amplified cell lines resistant to ALK tyrosine kinase inhibitors (TKIs) underwent apoptosis upon drug withdrawal as a consequence of ERK1/2 hyperactivation. Altogether, these findings indicate that an excess of NPM-ALK activation and signaling induces apoptosis via oncogenic stress responses. A ‘drug holiday’ where the ALK TKI treatment is suspended could represent a therapeutic option in cells that become resistant by NPM-ALK amplification.

Oncogenic JAK2V617F causes PD-L1 expression, mediating immune escape in myeloproliferative neoplasms

Oncogenic JAK mutation sensitizes myeloproliferative neoplasms to immune checkpoint inhibition. Cancers JAK up an immune checkpoint Myeloproliferative neoplasms, a group of hematologic cancers, are associated with mutations activating the JAK2 oncogene. JAK2 is located on chromosome 9, in the vicinity of the immunosuppressive PD-L1 gene, and Prestipino et al. discovered that myeloproliferative cancers with overactive JAK2 generally have increased PD-L1 as well. Although PD-L1 helps cancers evade the immune system, immune checkpoint inhibitors developed in recent years provide a way to block its function and turn PD-L1 expression into a therapeutic vulnerability for the tumors, as the authors demonstrate in this study. Recent evidence has revealed that oncogenic mutations may confer immune escape. A better understanding of how an oncogenic mutation affects immunosuppressive programmed death ligand 1 (PD-L1) expression may help in developing new therapeutic strategies. We show that oncogenic JAK2 (Janus kinase 2) activity caused STAT3 (signal transducer and activator of transcription 3) and STAT5 phosphorylation, which enhanced PD-L1 promoter activity and PD-L1 protein expression in JAK2V617F-mutant cells, whereas blockade of JAK2 reduced PD-L1 expression in myeloid JAK2V617F-mutant cells. PD-L1 expression was higher on primary cells isolated from patients with JAK2V617F–myeloproliferative neoplasms (MPNs) compared to healthy individuals and declined upon JAK2 inhibition. JAK2V617F mutational burden, pSTAT3, and PD-L1 expression were highest in primary MPN patient–derived monocytes, megakaryocytes, and platelets. PD-1 (programmed death receptor 1) inhibition prolonged survival in human MPN xenograft and primary murine MPN models. This effect was dependent on T cells. Mechanistically, PD-L1 surface expression in JAK2V617F-mutant cells affected metabolism and cell cycle progression of T cells. In summary, we report that in MPN, constitutive JAK2/STAT3/STAT5 activation, mainly in monocytes, megakaryocytes, and platelets, caused PD-L1–mediated immune escape by reducing T cell activation, metabolic activity, and cell cycle progression. The susceptibility of JAK2V617F-mutant MPN to PD-1 targeting paves the way for immunomodulatory approaches relying on PD-1 inhibition.

Oncogenic ALK regulates EMT in non-small cell lung carcinoma through repression of the epithelial splicing regulatory protein 1

A subset of Non-Small Cell Lung Carcinoma (NSCLC) carries chromosomal rearrangements involving the Anaplastic Lymphoma Kinase (ALK) gene. ALK-rearranged NSCLC are typically adenocarcinoma characterized by a solid signet-ring cell pattern that is frequently associated with a metastatic phenotype. Recent reports linked the presence of ALK rearrangement to an epithelial-mesenchymal transition (EMT) phenotype in NSCLC, but the extent and the mechanisms of an ALK-mediated EMT in ALK-rearranged NSCLC are largely unknown. We found that the ALK-rearranged H2228 and DFCI032, but not the H3122, cell lines displayed a mesenchymal phenotype. In these cell lines, oncogenic ALK activity dictated an EMT phenotype by directly suppressing E-cadherin and up-regulating vimentin expression, as well as expression of other genes involved in EMT. We found that the epithelial splicing regulatory protein 1 (ESRP1), a key regulator of the splicing switch during EMT, was repressed by EML4-ALK activity. The treatment of NSCLC cells with ALK tyrosine kinase inhibitors (TKIs) led to up-regulation of ESRP1 and E-cadherin, thus reverting the phenotype from mesenchymal to epithelial (MET). Consistently, ESRP1 knock-down impaired E-cadherin up-regulation upon ALK inhibition, whereas enforced expression of ESRP1 was sufficient to increase E-cadherin expression. These findings demonstrate an ALK oncogenic activity in the regulation of an EMT phenotype in a subset of NSCLC with potential implications for the biology of ALK-rearranged NSCLC in terms of metastatic propensity and resistance to therapy.

Current Immunotherapeutic Approaches in T Cell Non-Hodgkin Lymphomas

T cell non-Hodgkin lymphoma (T-NHL) is a rare and heterogeneous group of neoplasms of the lymphoid system. With the exception of a few relatively indolent entities, T-NHL is typically aggressive, treatment resistant, and associated with poor prognosis. Relatively few options with proven clinical benefit are available for patients with relapsed or refractory disease. Immunotherapy has emerged as a promising treatment for the management of patients with hematological malignancies. The identification of tumor antigens has provided a large number of potential targets. Therefore, several monoclonal antibodies (alemtuzumab, SGN-30, brentuximab vedotin, and mogamulizumab), directed against tumor antigens, have been investigated in different subtypes of T-NHL. In addition to targeting antigens involved in cancer cell physiology, antibodies can stimulate immune effector functions or counteract immunosuppressive mechanisms. Chimeric antigen receptor (CAR)-T cells directed against CD30 and immune checkpoint inhibitors are currently being investigated in clinical trials. In this review, we summarize the currently available clinical evidence for immunotherapy in T-NHL, focusing on the results of clinical trials using first generation monoclonal antibodies, new immunotherapeutic agents, immune checkpoint inhibitors, and CAR-T cell therapies.

Abstract 131: ALK oncogene regulates epithelial-mesenchymal transition (EMT) in ALK-rearranged non-small cell lung carcinoma through repression of the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2)

A subset of Non-Small Cell Lung Carcinoma (NSCLC) carries chromosomal rearrangements involving the Anaplastic Lymphoma Kinase (ALK) gene. More frequently ALK is juxtaposed to the echinoderm microtubule-associated protein-like 4 (EML4) gene on chromosome 2 and generates a constitutively active EML4-ALK fusion protein that triggers downstream oncogenic signals leading to increased cell proliferation and survival. The majority of ALK-rearranged NSCLC presents a peculiar histology characterized by a solid signet-ring cell and a mucinous cribriform pattern that is frequently associated with a metastatic phenotype. As the signet ring phenotype and metastasis are associated with epithelial-mesenchymal transition (EMT), a cellular reprogramming often activated in cancer cells during invasion and metastasis, we investigated whether ALK induces EMT in NSCLC. We performed RNA sequencing analysis on human ALK-rearranged NSCLC cell lines treated with ALK inhibitors or where EML4-ALK was knocked-down by shRNA. We found that EML-ALK regulated several genes related to EMT. In particular, the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2), key regulators of a splicing switch during EMT, were repressed in ALK-rearranged NSCLC cells and their repression was dependent on ALK activity. In keeping with these observations, H2228 and DFCI032 cells displayed a mesenchymal phenotype with almost complete suppression of the epithelial marker, E-cadherin, and a strong expression of the mesenchymal markers, vimentin and N-cadherin. In H2228 and DFCI032 cells, both E-cadherin suppression and vimentin up-regulation were dependent upon EML-ALK kinase activity because treatment with ALK inhibitors (TAE684 and crizotinib) or ALK knock-down reverted the phenotype of H2228 and DFCI032 from mesenchymal to epithelial and decreased their invasive potential. We excluded the involvement in EMT of ALK-rearranged NSCLC of other RTKs, such as EGFR or MET, because their inhibition did not have any effect on E-cadherin and vimentin expression. In ALK-rearranged NSCLC, the regulation of E-cadherin suppression was mainly transcriptional whereas vimentin regulation was post-transcriptional for both cell lines. Overexpression of ESRP1 led to up-regulation of E-cadherin, whereas ESRP1 knock-down impaired the reversion to an epithelial phenotype associated to inhibition of ALK activity. In conclusion, we showed that oncogenic ALK regulates EMT in NSCLC through ESRP repression. These findings could have implications for the biology of ALK-rearranged NSCLC in terms of metastatic potential and resistance to therapy. Citation Format: Claudia Voena, Lydia Varesio, Liye Zhang, Matteo Menotti, Teresa Poggio, Filomena Di Giacomo, Elena Panizza, Cristina Mastini, Mara Compagno, Stefano Monti, Roberto Chiarle. ALK oncogene regulates epithelial-mesenchymal transition (EMT) in ALK-rearranged non-small cell lung carcinoma through repression of the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 131. doi:10.1158/1538-7445.AM2015-131