Lenalidomide enhances CD23.CAR T cell therapy in chronic lymphocytic leukemia.

Chimeric antigen receptors (CAR)-modified T cells are an emerging therapeutic tool for chronic lymphocytic leukemia (CLL). However, in patients with CLL, well-known T-cell defects and the inhibitory properties of the tumor microenvironment (TME) hinder the efficacy of CAR T cells. We explored a novel approach combining CARs with lenalidomide, an immunomodulatory drug that tempers the immunosuppressive activity of the CLL TME. T cells from patients with CLL were engineered to express a CAR specific for CD23, a promising target antigen. Lenalidomide maintained the in vitro effector functions of CD23.CAR+ T cells effector functions in terms of antigen-specific cytotoxicity, cytokine release and proliferation. Overall, lenalidomide preserved functional CAR T-CLL cell immune synapses. In a Rag2-/-γc-/--based xenograft model of CLL, we demonstrated that, when combined with low-dose lenalidomide, CD23.CAR+ T cells efficiently migrated to leukemic sites and delayed disease progression when compared to CD23.CAR+ T cells given with rhIL-2. These observations underline the therapeutic potential of this novel CAR-based combination strategy in CLL.

Trabectedin Reveals a Strategy of Immunomodulation in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a B-cell neoplasia characterized by protumor immune dysregulation involving nonmalignant cells of the microenvironment, including T lymphocytes and tumor-associated myeloid cells. Although therapeutic agents have improved treatment options for CLL, many patients still fail to respond. Some patients also show immunosuppression. We have investigated trabectedin, a marine-derived compound with cytotoxic activity on macrophages in solid tumors. Here, we demonstrate that trabectedin induces apoptosis of human primary leukemic cells and also selected myeloid and lymphoid immunosuppressive cells, mainly through the TRAIL/TNF pathway. Trabectedin modulates transcription and translation of IL6, CCL2, and IFNα in myeloid cells and FOXP3 in regulatory T cells. Human memory CD8+ T cells downregulate PD-1 and, along with monocytes, exert in vivo antitumor function. In xenograft and immunocompetent CLL mouse models, trabectedin has antileukemic effects and antitumor impact on the myeloid and lymphoid cells compartment. It depletes myeloid-derived suppressor cells and tumor-associated macrophages and increases memory T cells. Trabectedin also blocks the PD-1/PD-L1 axis by targeting PD-L1+ CLL cells, PD-L1+ monocytes/macrophages, and PD-1+ T cells. Thus, trabectedin behaves as an immunomodulatory drug with potentially attractive therapeutic value in the subversion of the protumor microenvironment and in overcoming chemoimmune resistance.

Olaptesed pegol (NOX-A12) with bendamustine and rituximab: a phase IIa study in patients with relapsed/refractory chronic lymphocytic leukemia.

Olaptesed pegol (NOX-A12) is a pegylated structured L-oligoribonucleotide that binds and neutralizes CXCL12, a chemokine tightly regulating the life cycle of chronic lymphocytic leukemia cells. The resulting inhibition of CXCR4 and CXCR7 signaling reduces the protective activity of the bone marrow and lymph node microenvironment. CXCL12 inhibition mobilizes chronic lymphocytic leukemia cells into the circulation and prevents their homing into the protective niches. In this phase I/II study, 28 patients with relapsed/refractory chronic lymphocytic leukemia were treated with olaptesed pegol in combination with bendamustine and rituximab. Combination treatment was preceded by single escalating pilot doses of olaptesed pegol in the first ten patients for evaluation of safety and pharmacokinetics. Peak concentrations and systemic exposure of olaptesed pegol were dose-linear; plasma elimination was monophasic with a 53.2 h half-life. A rapid increase in circulating chronic lymphocytic leukemia cells was observed already 1 h after administration of olaptesed pegol and lasted for at least 72 h. Single-agent treatment was well tolerated and no dose-limiting toxicity was observed. The combination regimen yielded an overall response rate of 86%, with 11% of patients achieving a complete response and 75% a partial response. Notably, all ten high-risk patients, including four with a 17p deletion, responded to treatment. The median progression-free survival was 15.4 (95% confidence interval: 12.2, 26.2) months while the median overall survival was not reached with >80% of patients alive after a median follow-up of 28 months. Olaptesed pegol was well tolerated and did not result in additional toxicity when combined with bendamustine and rituximab (ClinicalTrials.gov identifier: NCT01486797). Further clinical development of this novel CXCL12 inhibitor is thus warranted.

IL1R8 Deficiency Drives Autoimmunity-Associated Lymphoma Development.

Chronic inflammation, including that driven by autoimmunity, is associated with the development of B-cell lymphomas. IL1R8 is a regulatory receptor belonging to the IL1R family, which negatively regulates NF-κB activation following stimulation of IL1R or Toll-like receptor family members. IL1R8 deficiency is associated with the development of severe autoimmune lupus-like disease in lpr mice. We herein investigated whether concomitant exacerbated inflammation and autoimmunity caused by the deficiency of IL1R8 could recapitulate autoimmunity-associated lymphomagenesis. We thus monitored B-cell lymphoma development during the aging of IL1R8-deficient lpr mice, observing an increased lymphoid cell expansion that evolved to diffuse large B-cell lymphoma (DLBCL). Molecular and gene-expression analyses showed that the NF-κB pathway was constitutively activated in Il1r8 -/-/lpr B splenocytes. In human DLBCL, IL1R8 had reduced expression compared with normal B cells, and higher IL1R8 expression was associated with a better outcome. Thus, IL1R8 silencing is associated with increased lymphoproliferation and transformation in the pathogenesis of B-cell lymphomas associated with autoimmunity.

A retinoic acid-dependent stroma-leukemia crosstalk promotes chronic lymphocytic leukemia progression.

In chronic lymphocytic leukemia (CLL), the non-hematopoietic stromal microenvironment plays a critical role in promoting tumor cell recruitment, activation, survival, and expansion. However, the nature of the stromal cells and molecular pathways involved remain largely unknown. Here, we demonstrate that leukemic B lymphocytes induce the activation of retinoid acid synthesis and signaling in the microenvironment. Inhibition of RA-signaling in stromal cells causes deregulation of genes associated with adhesion, tissue organization and chemokine secretion including the B-cell chemokine CXCL13. Notably, reducing retinoic acid precursors from the diet or inhibiting RA-signaling through retinoid-antagonist therapy prolong survival by preventing dissemination of leukemia cells into lymphoid tissues. Furthermore, mouse and human leukemia cells could be distinguished from normal B-cells by their increased expression of Rarγ2 and RXRα, respectively. These findings establish a role for retinoids in murine CLL pathogenesis, and provide new therapeutic strategies to target the microenvironment and to control disease progression.

iwCLL guidelines for diagnosis, indications for treatment, response assessment, and supportive management of CLL.

The previous edition of the consensus guidelines of the International Workshop on Chronic Lymphocytic Leukemia (iwCLL), published in 2008, has found broad acceptance by physicians and investigators caring for patients with CLL. Recent advances including the discovery of the genomic landscape of the disease, the development of genetic tests with prognostic relevance, and the detection of minimal residual disease (MRD), coupled with the increased availability of novel targeted agents with impressive efficacy, prompted an international panel to provide updated evidence- and expert opinion-based recommendations. These recommendations include a revised version of the iwCLL response criteria, an update on the use of MRD status for clinical evaluation, and recommendations regarding the assessment and prophylaxis of viral diseases during management of CLL.

Modeling multiple myeloma-bone marrow interactions and response to drugs in a 3D surrogate microenvironment.

Multiple myeloma develops primarily inside the bone marrow microenvironment, that confers pro-survival signals and drug resistance. 3D cultures that reproduce multiple myeloma-bone marrow interactions are needed to fully investigate multiple myeloma pathogenesis and response to drugs. To this purpose, we exploited the 3D Rotary Cell Culture System bioreactor technology for myeloma-bone marrow co-cultures in gelatin scaffolds. The model was validated with myeloma cell lines that, as assessed by histochemical and electron-microscopic analyses, engaged contacts with stromal cells and endothelial cells. Consistently, pro-survival signaling and also cell adhesion-mediated drug resistance were significantly higher in 3D than in 2D parallel co-cultures. The contribution of the VLA-4/VCAM1 pathway to resistance to bortezomib was modeled by the use of VCAM1 transfectants. Soluble factor-mediated drug resistance could be also demonstrated in both 2D and 3D co-cultures. The system was then successfully applied to co-cultures of primary myeloma cells-primary myeloma bone marrow stromal cells from patients and endothelial cells, allowing the development of functional myeloma-stroma interactions and MM cell long-term survival. Significantly, genomic analysis performed in a high-risk myeloma patient demonstrated that culture in bioreactor paralleled the expansion of the clone that ultimately dominated in vivo Finally, the impact of bortezomib on myeloma cells and on specialized functions of the microenvironment could be evaluated. Our findings indicate that 3D dynamic culture of reconstructed human multiple myeloma microenvironments in bioreactor may represent a useful platform for drug testing and for studying tumor-stroma molecular interactions.

Pathogen-specific B-cell receptors drive chronic lymphocytic leukemia by light-chain-dependent cross-reaction with autoantigens.

Several lines of evidence indirectly suggest that antigenic stimulation through the B-cell receptor (BCR) supports chronic lymphocytic leukemia (CLL) development. In addition to self-antigens, a number of microbial antigens have been proposed to contribute to the selection of the immunoglobulins expressed in CLL. How pathogen-specific BCRs drive CLL development remains, however, largely unexplored. Here, we utilized mouse models of CLL pathogenesis to equip B cells with virus-specific BCRs and study the effect of antigen recognition on leukemia growth. Our results show that BCR engagement is absolutely required for CLL development. Unexpectedly, however, neither acute nor chronic exposure to virus-derived antigens influenced leukemia progression. Rather, CLL clones preferentially selected light chains that, when paired with virus-specific heavy chains, conferred B cells the ability to recognize a broad range of autoantigens. Taken together, our results suggest that pathogens may drive CLL pathogenesis by selecting and expanding pathogen-specific B cells that cross-react with one or more self-antigens.

Invariant NKT cells contribute to chronic lymphocytic leukemia surveillance and prognosis.

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant CD5+ B lymphocytes in blood, bone marrow, and lymphoid organs. CD1d-restricted invariant natural killer T (iNKT) cells are innate-like T lymphocytes strongly implicated in tumor surveillance. We investigated the impact of iNKT cells in the natural history of the disease in the Eµ-Tcl1 (Tcl1) CLL mouse model and 68 CLL patients. We found that Tcl1-CLL cells express CD1d and that iNKT cells critically delay disease onset but become functionally impaired upon disease progression. In patients, disease progression correlates with high CD1d expression on CLL cells and impaired iNKT cells. Conversely, disease stability correlates with negative or low CD1d expression on CLL cells and normal iNKT cells, suggesting indirect leukemia control. iNKT cells indeed hinder CLL survival in vitro by restraining CD1d-expressing nurse-like cells, a relevant proleukemia macrophage population. Multivariable analysis identified iNKT cell frequency as an independent predictor of disease progression. Together, these results support the contribution of iNKT cells to CLL immune surveillance and highlight iNKT cell frequency as a prognostic marker for disease progression.

Functional Differences between IgM and IgD Signaling in Chronic Lymphocytic Leukemia.

BCR signaling is a central pathogenetic pathway in chronic lymphocytic leukemia (CLL). Most CLL cells express BCRs of IgM and IgD isotypes, but the contribution of these isotypes to functional responses remains incompletely defined. We therefore investigated differences between IgM and IgD signaling in freshly isolated peripheral blood CLL cells and in CLL cells cultured with nurselike cells, a model that mimics the lymph node microenvironment. IgM signaling induced prolonged activation of ERK kinases and promoted CLL cell survival, CCL3 and CCL4 chemokine secretion, and downregulation of BCL6, the transcriptional repressor of CCL3 In contrast, IgD signaling induced activation of the cytoskeletal protein HS1, along with F-actin polymerization, which resulted in rapid receptor internalization and failure to support downstream responses, including CLL cell survival and chemokine secretion. IgM and IgD receptor downmodulation, HS1 and ERK activation, chemokine secretion, and BCL6 downregulation were also observed when CLL cells were cocultured with nurselike cells. The Bruton's tyrosine kinase inhibitor ibrutinib effectively inhibited both IgM and IgD isotype signaling. In conclusion, through a variety of functional readouts, we demonstrate very distinct outcomes of IgM and IgD isotype activation in CLL cells, providing novel insight into the regulation of BCR signaling in CLL.

Inhibition of chronic lymphocytic leukemia progression by full-length chromogranin A and its N-terminal fragment in mouse models.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of leukemic B cells in peripheral blood, bone marrow (BM) and lymphoid tissues, and by their recirculation between these compartments. We observed that circulating chromogranin A (CgA) and its N-terminal fragment (called vasostatin-1, CgA1-76), two neuroendocrine secretory polypeptides that enhance the endothelial barrier function, are present in variable amounts in the blood of CLL patients. Studies in animal models showed that daily administration of full-length human CgA1-439 (0.3 µg, i.v., or 1.5 µg/mouse, i.p.) can reduce the BM/blood ratio of leukemic cells in Eµ-TCL1 mice, a transgenic model, and decrease BM, lung and kidney infiltration in Rag2-/-γc-/- mice engrafted with human MEC1 CLL cells, a xenograft model. This treatment also reduced the loss of body weight and improved animal motility. In vitro, CgA enhanced the endothelial barrier integrity and the trans-endothelial migration of MEC1 cells, with a bimodal dose-response curve. Vasostatin-1, but not a larger fragment consisting of N-terminal and central regions of CgA (CgA1-373), inhibited CLL progression in the xenograft model, suggesting that the C-terminal region is crucial for CgA activity and that the N-terminal domain contains a site that is activated by proteolytic cleavage. These findings suggest that circulating full-length CgA and its fragments may contribute to regulate leukemic cell trafficking and reduce tissue infiltration in CLL.

Targeting Macrophages Sensitizes Chronic Lymphocytic Leukemia to Apoptosis and Inhibits Disease Progression.

The role of monocytes/macrophages in the development and progression of chronic lymphocytic leukemia (CLL) is poorly understood. Transcriptomic analyses show that monocytes/macrophages and leukemic cells cross talk during CLL progression. Macrophage depletion impairs CLL engraftment, drastically reduces leukemic growth, and favorably impacts mouse survival. Targeting of macrophages by either CSF1R signaling blockade or clodrolip-mediated cell killing has marked inhibitory effects on established leukemia also. Macrophage killing induces leukemic cell death mainly via the TNF pathway and reprograms the tumor microenvironment toward an antitumoral phenotype. CSF1R inhibition reduces leukemic cell load, especially in the bone marrow, and increases circulating CD20(+) leukemic cells. Accordingly, co-targeting TAMs and CD20-expressing leukemic cells provides a survival benefit in the mice. These results establish the important role of macrophages in CLL and suggest therapeutic strategies based on interfering with leukemia-macrophage interactions.

Chromogranin A Is Preferentially Cleaved into Proangiogenic Peptides in the Bone Marrow of Multiple Myeloma Patients.

Angiogenesis has been postulated to be critical for the pathogenesis of multiple myeloma, a neoplastic disease characterized by abnormal proliferation of malignant plasma cells in the bone marrow (BM). Cleavage of the N- and C-terminal regions of circulating chromogranin A (CgA, CHGA), classically an antiangiogenic protein, can activate latent antiangiogenic and proangiogenic sites, respectively. In this study, we investigated the distribution of CgA-derived polypeptides in multiple myeloma patients and the subsequent implications for disease progression. We show that the ratio of pro/antiangiogenic forms of CgA is altered in multiple myeloma patients compared with healthy subjects and that this ratio is higher in BM plasma compared with peripheral plasma, suggesting enhanced local cleavage of the CgA C-terminal region. Enhanced cleavage correlated with increased VEGF and FGF2 BM plasma levels and BM microvascular density. Using the Vk*MYC mouse model of multiple myeloma, we further demonstrate that exogenously administered CgA was cleaved in favor of the proangiogenic form and was associated with increased microvessel density. Mechanistic studies revealed that multiple myeloma and proliferating endothelial cells can promote CgA C-terminal cleavage by activating the plasminogen activator/plasmin system. Moreover, cleaved and full-length forms could also counter balance the pro/antiangiogenic activity of each other in in vitro angiogenesis assays. These findings suggest that the CgA-angiogenic switch is activated in the BM of multiple myeloma patients and prompt further investigation of this CgA imbalance as a prognostic or therapeutic target. Cancer Res; 76(7); 1781-91. ©2016 AACR.

HIF-1α regulates the interaction of chronic lymphocytic leukemia cells with the tumor microenvironment.

Hypoxia-inducible transcription factors (HIFs) regulate a wide array of adaptive responses to hypoxia and are often activated in solid tumors and hematologic malignancies due to intratumoral hypoxia and emerging new layers of regulation. We found that in chronic lymphocytic leukemia (CLL), HIF-1α is a novel regulator of the interaction of CLL cells with protective leukemia microenvironments and, in turn, is regulated by this interaction in a positive feedback loop that promotes leukemia survival and propagation. Through unbiased microarray analysis, we found that in CLL cells, HIF-1α regulates the expression of important chemokine receptors and cell adhesion molecules that control the interaction of leukemic cells with bone marrow and spleen microenvironments. Inactivation of HIF-1α impairs chemotaxis and cell adhesion to stroma, reduces bone marrow and spleen colonization in xenograft and allograft CLL mouse models, and prolongs survival in mice. Of interest, we found that in CLL cells, HIF-1α is transcriptionally regulated after coculture with stromal cells. Furthermore, HIF-1α messenger RNA levels vary significantly within CLL patients and correlate with the expression of HIF-1α target genes, including CXCR4, thus further emphasizing the relevance of HIF-1α expression to CLL pathogenesis.

High Doses of Antimetabolites Followed by High-Dose Sequential Chemoimmunotherapy and Autologous Stem-Cell Transplantation in Patients With Systemic B-Cell Lymphoma and Secondary CNS Involvement: Final Results of a Multicenter Phase II Trial.

PURPOSE: Treatment of secondary CNS dissemination in patients with aggressive lymphomas remains an important, unmet clinical need. Herein, we report the final results of a multicenter phase II trial addressing a new treatment for secondary CNS lymphoma based on encouraging experiences with high doses of antimetabolites in primary CNS lymphoma and with rituximab plus high-dose sequential chemoimmunotherapy (R-HDS) in relapsed aggressive lymphoma. PATIENTS AND METHODS: HIV-negative patients with aggressive B-cell lymphoma and secondary CNS involvement at diagnosis or relapse, age 18 to 70 years, and Eastern Cooperative Oncology Group performance status ≤ 3 were enrolled and treated with high-doses of methotrexate and cytarabine, followed by R-HDS (cyclophosphamide, cytarabine, and etoposide) supported by autologous stem-cell transplantation (ASCT). Treatment included eight doses of rituximab and four doses of intrathecal liposomal cytarabine. The primary end point was 2-year event-free survival; the planned accrual was 38 patients. RESULTS: Thirty-eight patients were enrolled; CNS disease was detected at presentation in 16 patients. Toxicity was usually hematologic and manageable, with grade 4 febrile neutropenia in 3% of delivered courses and grade 4 nonhematologic toxicity in 2% of delivered courses. Four patients died because of toxicity. Autologous stem cells were successfully collected in 24 (89%) of 27 patients (median, 10 × 10(6)/kg); 20 patients underwent ASCT. Complete response was achieved in 24 patients (complete response rate, 63%; 95% CI, 48% to 78%). At a median follow-up of 48 months, 17 patients remained relapse free, with a 2-year event-free survival rate of 50% ± 8%. At 5 years, 16 patients were alive, with a 5-year overall survival rate of 41% ± 8% for the whole series and 68% ± 11% for patients who received transplantation. Systemic (extra-CNS) and/or meningeal disease did not affect outcome. CONCLUSION: The combination of high doses of antimetabolites, R-HDS, and ASCT is feasible and effective in patients age 18 to 70 years old with secondary CNS lymphoma, and we propose it as a new standard therapeutic option.

Toll-like receptor stimulation in splenic marginal zone lymphoma can modulate cell signaling, activation and proliferation.

Recent studies on splenic marginal zone lymphoma identified distinct mutations in genes belonging to the B-cell receptor and Toll-like receptor signaling pathways, thus pointing to their potential implication in the biology of the disease. However, limited data is available regarding the exact role of TLRs. We aimed at characterizing the expression pattern of TLRs in splenic marginal zone lymphoma cells and their functional impact on the activation, proliferation and viability of malignant cells in vitro. Cells expressed significant levels of TLR1, TLR6, TLR7, TLR8, TLR9 and TLR10 mRNA; TLR2 and TLR4 showed a low, variable pattern of expression among patients whereas TLR3 and TLR5 mRNAs were undetectable; mRNA specific for TLR signaling molecules and adapters was also expressed. At the protein level, TLR1, TLR6, TLR7, TLR9 and TLR10 were detected. Stimulation of TLR1/2, TLR2/6 and TLR9 with their respective ligands triggered the activation of IRAK kinases, MAPK and NF-κB signaling pathways, and the induction of CD86 and CD25 activation molecules, although in a heterogeneous manner among different patient samples. TLR-induced activation and cell viability were also inhibited by a specific IRAK1/4 inhibitor, thus strongly supporting the specific role of TLR signaling in these processes. Furthermore, TLR2/6 and TLR9 stimulation also significantly increased cell proliferation. In conclusion, we demonstrate that splenic marginal zone lymphoma cells are equipped with functional TLR and signaling molecules and that the stimulation of TLR1/2, TLR2/6 and TLR9 may play a role in regulating disease pathobiology, likely promoting the expansion of the neoplastic clone.

Establishment and Characterization of PCL12, a Novel CD5+ Chronic Lymphocytic Leukaemia Cell Line.

Immortalized cell lines representative of chronic lymphocytic leukemia (CLL) can assist in understanding disease pathogenesis and testing new therapeutic agents. At present, very few representative cell lines are available. We here describe the characterization of a new cell line (PCL12) that grew spontaneously from the peripheral blood (PB) of a CLL patient with progressive disease and EBV infection. The CLL cell origin of PCL12 was confirmed after the alignment of its IGH sequence against the "original" clonotypic sequence. The IGH gene rearrangement was truly unmutated and no CLL-related cytogenetic or genetic lesions were detected. PCL12 cells express CD19, CD20, CD5, CD23, low levels of IgM and IgD and the poor-outcome-associated prognostic markers CD38, ZAP70 and TCL1. In accordance with its aggressive phenotype the cell line is inactive in terms of LYN and HS1 phosphorylation. BcR signalling pathway is constitutively active and anergic in terms of p-ERK and Calcium flux response to α-IgM stimulation. PCL12 cells strongly migrate in vitro in response to SDF-1 and form clusters. Finally, they grow rapidly and localize in all lymphoid organs when xenotrasplanted in Rag2-/-γc-/- mice. PCL12 represents a suitable preclinical model for testing pharmacological agents.