Cinzia Bologna

Targeting the microenvironment in chronic lymphocytic leukemia offers novel therapeutic options.

Chronic lymphocytic leukemia (CLL) cells display features consistent with a defect in apoptosis and exhibit prolonged survival in vivo. Survival of these malignant cells is influenced by interactions with non-leukemic cells located in permissive niches in lymphoid organs. Leukemic cells subvert the normal architecture of the lymphoid organs, recruiting stromal cells, dendritic cells and T lymphocytes, all reported as playing active roles in the survival and proliferation of CLL. The same survival-promoting environment also rescues/protects leukemic cells from cytotoxic therapies, giving way to disease relapse. This review summarizes and discusses current knowledge about the intricate network of soluble and cell-bound signals regulating the life and death of CLL cells in different districts. At the same time, it seeks to hone in on which discrete molecular elements are best suited as targets for treating this still incurable disease.

NAD+-metabolizing ecto-enzymes shape tumor-host interactions: the chronic lymphocytic leukemia model.

Nicotinamide adenine dinucleotide (NAD+) is an essential co‐enzyme that can be released in the extracellular milieu. Here, it may elicit signals through binding purinergic receptors. Alternatively, NAD+ may be dismantled to adenosine, up‐taken by cells and transformed to reconstitute the intracellular nucleotide pool. An articulated ecto‐enzyme network is responsible for the nucleotide–nucleoside conversion. CD38 is the main mammalian enzyme that hydrolyzes NAD+, generating Ca2+‐active metabolites. Evidence suggests that this extracellular network may be altered or used by tumor cells to (i) nestle in protected areas, and (ii) evade the immune response. We have exploited chronic lymphocytic leukemia as a model to test the role of the ecto‐enzyme network, starting by analyzing the individual elements that make up the whole picture.

PD-L1 up-regulation in melanoma increases disease aggressiveness and is mediated through miR-17-5p

PD-L1 is expressed by a subset of patients with metastatic melanoma (MM) with an unfavorable outcome. Its expression is increased in cells resistant to BRAF or MEK inhibitors (BRAFi or MEKi). However, the function and regulation of expression of PD-L1 remain incompletely understood. After generating BRAFi- and MEKi-resistant cell lines, we observed marked up-regulation of PD-L1 expression. These cells were characterized by a common gene expression profile with up-regulation of genes involved in cell movement. Consistently, in vitro they showed significantly increased invasive properties. This phenotype was controlled in part by PD-L1, as determined after silencing the molecule. Up-regulation of PD-L1 was due to post-transcriptional events controlled by miR-17-5p, which showed an inverse correlation with PD-L1 mRNA. Direct binding between miR-17-5p and the 3’-UTR of PD-L1 mRNA was demonstrated using luciferase reporter assays. In a cohort of 80 BRAF-mutated MM patients treated with BRAFi or MEKi, constitutive expression of PD-L1 in the absence of immune infiltrate, defined the patient subset with the worst prognosis. Furthermore, PD-L1 expression increased in tissue biopsies after the metastatic lesions became resistant to BRAFi or MEKi. Lastly, plasmatic miR-17-5p levels were higher in patients with PD-L1+ than PD-L1- lesions. In conclusion, our findings indicate that PD-L1 expression induces a more aggressive behavior in melanoma cells. We also show that PD-L1 up-regulation in BRAFi or MEKi-resistant cells is partly due to post-transcriptional mechanisms that involve miR-17-5p, suggesting that miR-17-5p may be used as a marker of PD-L1 expression by metastatic lesions and ultimately a predictor of responses to BRAFi or MEKi.

Mutations in NOTCH1 PEST domain orchestrate CCL19-driven homing of chronic lymphocytic leukemia cells by modulating the tumor suppressor gene DUSP22

Even if NOTCH1 is commonly mutated in chronic lymphocytic leukemia (CLL), its functional impact in the disease remains unclear. Using CRISPR/Cas9-generated Mec-1 cell line models, we show that NOTCH1 regulates growth and homing of CLL cells by dictating expression levels of the tumor suppressor gene DUSP22. Specifically, NOTCH1 affects the methylation of DUSP22 promoter by modulating a nuclear complex, which tunes the activity of DNA methyltransferase 3A (DNMT3A). These effects are enhanced by PEST-domain mutations, which stabilize the molecule and prolong signaling. CLL patients with a NOTCH1-mutated clone showed low levels of DUSP22 and active chemotaxis to CCL19. Lastly, in xenograft models, NOTCH1-mutated cells displayed a unique homing behavior, localizing preferentially to the spleen and brain. These findings connect NOTCH1, DUSP22, and CCL19-driven chemotaxis within a single functional network, suggesting that modulation of the homing process may provide a relevant contribution to the unfavorable prognosis associated with NOTCH1 mutations in CLL.

SLAMF1 regulation of chemotaxis and autophagy determines CLL patient response.

Chronic lymphocytic leukemia (CLL) is a variable disease; therefore, markers to identify aggressive forms are essential for patient management. Here, we have shown that expression of the costimulatory molecule and microbial sensor SLAMF1 (also known as CD150) is lost in a subset of patients with an aggressive CLL that associates with a shorter time to first treatment and reduced overall survival. SLAMF1 silencing in CLL-like Mec-1 cells, which constitutively express SLAMF1, modulated pathways related to cell migration, cytoskeletal organization, and intracellular vesicle formation and recirculation. SLAMF1 deficiency associated with increased expression of CXCR4, CD38, and CD44, thereby positively affecting chemotactic responses to CXCL12. SLAMF1 ligation with an agonistic monoclonal antibody increased ROS accumulation and induced phosphorylation of p38, JNK1/2, and BCL2, thereby promoting the autophagic flux. Beclin1 dissociated from BCL2 in response to SLAMF1 ligation, resulting in formation of the autophagy macrocomplex, which contains SLAMF1, beclin1, and the enzyme VPS34. Accordingly, SLAMF1-silenced cells or SLAMF1(lo) primary CLL cells were resistant to autophagy-activating therapeutic agents, such as fludarabine and the BCL2 homology domain 3 mimetic ABT-737. Together, these results indicate that loss of SLAMF1 expression in CLL modulates genetic pathways that regulate chemotaxis and autophagy and that potentially affect drug responses, and suggest that these effects underlie unfavorable clinical outcome experienced by SLAMF1(lo) patients.

CD38 in chronic lymphocytic leukemia: from bench to bedside?

Human CD38 is a cell surface molecule endowed with multiple functions. As an enzyme, it catalyzes the production of Ca2+ active metabolites, predominantly cADPR and ADPR. As a receptor, it regulates the activation of an intracellular signaling pathway, generally linked to lymphocyte activation and proliferation in physiological conditions. The finding that CD38 behaves as an independent negative prognostic factor in CLL patients was the starting point for investigations into the functional role of the molecule in the neoplastic context. Data accumulating in over a decade concur to define a model where CD38 is a central element of a large supramolecular complex that includes surface signaling receptors, chemokine receptors, adhesion molecules and matrix metalloproteases. Expression of CD38 within this supramolecular complex makes signal transduction as well as chemotaxis and homing more efficient, suggesting that the molecule is an integrator of proliferative and migratory signals. These data indicate that CD38 is not only a reliable disease marker but also a functional molecule in the CLL context. The next decade will likely tell whether it can also be a useful therapeutic target.

Adenosine signaling mediates hypoxic responses in the chronic lymphocytic leukemia microenvironment

The chronic lymphocytic leukemia (CLL) niche is a closed environment where leukemic cells derive growth and survival signals through their interaction with macrophages and T lymphocytes. Here, we show that the CLL lymph node niche is characterized by overexpression and activation of HIF-1α, which increases adenosine generation and signaling, affecting tumor and host cellular responses. Hypoxia in CLL lymphocytes modifies central metabolic pathways, protects against drug-driven apoptosis, and induces interleukin 10 (IL-10) production. In myeloid cells, it forces monocyte differentiation to macrophages expressing IRF4, IDO, CD163, and CD206, hallmarks of the M2 phenotype, which promotes tumor progression. It also induces IL-6 production and enhances nurturing properties. Low oxygen levels decrease T-cell proliferation, promote glycolysis, and cause the appearance of a population of PD-1+ and IL-10-secreting T cells. Blockade of the A2A adenosine receptor counteracts these effects on all cell populations, making leukemic cells more susceptible to pharmacological agents while restoring immune competence and T-cell proliferation. Together, these results indicate that adenosine signaling through the A2A receptor mediates part of the effects of hypoxia. They also suggest that therapeutic strategies to inhibit the adenosinergic axis may be useful adjuncts to chemotherapy or tyrosine kinase inhibitors in the treatment of CLL patients.

Abstract 5583: Pegylated adenosine deaminase 2 (PEG-ADA2) abrogates the cytoprotective effects of adenosine against chronic lymphocytic leukemia cells

Extracellular adenosine can be generated from ATP and/or ADP through the concerted action of the ectoenzymes CD39 and CD73. Adenosine can bind different type-1 purinergic receptors eliciting potent cytoprotective and immunosuppressive effects. We previously focused on chronic lymphocytic leukemia (CLL), a disease characterized by the progressive expansion of a mature population of B lymphocytes, showing that a subset of patients with a CD73+ clone possess a poor prognosis. This patient subset can actively generate adenosine, which favors leukemic cell survival, synthesis of immunosuppressive cytokines, while inhibiting T lymphocyte proliferation. ADA2 is a human enzyme that catalyzes the conversion of adenosine into inosine. We hypothesized that depletion of high adenosine levels from the tumor microenvironment through the administration of exogenous ADA2 could be therapeutically relevant to limit tumor protection and immunosuppression. Several engineered PEG-ADA2 variants possessed improved enzyme activity and pharmacokinetics compared to wild-type ADA2. Inhibition of adenosine receptor activation was evaluated by measuring intracellular cAMP concentrations in T lymphocytes purified from healthy donors. Adenosine depletion was observed with increasing concentrations of PEG-ADA2-R222Q/S265N and significantly reduced cAMP levels. PEG-ADA2 variants were then tested on CD73+ CLL lymphocytes to determine their effects on extracellular adenosine and inosine. Treatment of leukemic B cells with PEG-ADA2-R222Q/S265N and PEG-ADA2-R222Q/S265N/K374D resulted in complete removal of high levels of adenosine, as measured by HPLC. Conversely, PEG-ADA2-E182T, which possesses significantly attenuated catalytic activity, was less effective in reducing adenosine. The functional effects of adenosine depletion on CLL cell survival were analyzed following treatment with the chemotherapeutic, DNA-damaging agent etoposide that robustly induces apoptosis within 16 hours. Addition of exogenous adenosine to cultures of purified CD73+ CLL cells significantly rescued cells from etoposide-induced apoptosis. However, when these primary leukemic cells were pretreated with PEG-ADA2-R222Q/S265N or PEG-ADA2-R222Q/S265N/K374D, the cell viability rate was significantly decreased, abrogating the cytoprotective effects of adenosine. On the contrary, PEG-ADA2-E182T had a minimal effect, suggesting enzymatic depletion of adenosine is critical to observe these effects. Similar effects were observed by the PEG-ADA2 variants on a CLL cell line expressing CD73+. These preliminary results suggest that enzymatic depletion of extracellular adenosine following treatment with PEG-ADA2 is a relevant approach to counteract the cytoprotective effects of adenosine, warranting further development of PEG-ADA2 as a possible approach to treat CLL. Citation Format: Sara Serra, Cinzia Bologna, Luz Londono, Lin Wang, Michael Shepard, Sanna Rosengren, Christopher Thanos, Silvia Deaglio. Pegylated adenosine deaminase 2 (PEG-ADA2) abrogates the cytoprotective effects of adenosine against chronic lymphocytic leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5583. doi:10.1158/1538-7445.AM2017-5583

Nicotinamide Phosphoribosyltransferase (NAMPT) as a Therapeutic Target in BRAF-Mutated Metastatic Melanoma

Background One of the effects of oncogenic signaling is metabolic reprogramming of tumor cells to support anabolic growth, opening the way to therapeutic targeting of metabolic pathways. Methods We studied NAD biosynthesis in BRAF inhibitor (BRAFi)-resistant (BiR) melanoma cell lines. Data in cell lines were confirmed by immunohistochemistry in biopsies from 17 patients with metastatic melanoma (MM) before and after the acquisition of resistance to BRAFi. Therapeutic potential of NAD biosynthesis inhibitors was determined by invitro monitoring cell growth and death and in mouse xenograft models. Mice (n = 6-10 mice/group) were treated with nicotinamide phosphoribosyltranferase inhibitor (NAMPTi), BRAFi, or their combination, and tumor growth and survival were analyzed. All statistical tests were two-sided. Results BiR cells had higher NAD levels compared with their BRAFi-sensitive counterparts (P < .001 and P = .001 for M14 and A375, respectively) and with normal melanocytes (P < .001), achieved through transcriptional upregulation of the enzyme NAMPT, which became the master regulator of NAD synthesis. Conversely, treatment with BRAFi or MEK inhibitors decreased NAMPT expression and cellular NAD levels. Robust NAMPT upregulation was documented in tissue biopsies from MM patients after development of resistance to BRAFi (P < .001). Treatment of melanoma cells with NAMPTi depleted NAD and ATP, depolarized mitochondrial membrane, and led to reactive oxygen species production, blocking cells in the G2/M phase and inducing apoptosis. Treatment of BiR xenografts with NAMPTi improved mouse survival (median survival of vehicle-treated mice was 52 days vs 100 days for NAMPTi-treated ones in M14/BiR, while in A375/BiR median survival of vehicle-treated mice was 23.5 days vs 43 days for NAMPTi-treated ones, P < .001). Conclusions BiR melanoma cells overexpress NAMPT, which acts as a connecting element between BRAF oncogenic signaling and metabolism, becoming an actionable target for this subset of MM patients.

Linking SLAMF1 to autophagy and sensitivity to therapy in chronic lymphocytic leukemia

ABSTRACT We recently reported that expression of the costimulatory molecule and microbial sensor SLAMF1 (signaling lymphocytic activation molecule family 1, also known as CD150) is lost in chronic lymphocytic leukemia (CLL) patients characterized by a shorter overall survival. SLAMF1 modulates CLL responses to chemokines and regulates autophagy. Loss of SLAMF1 renders CLL cells relatively unresponsive to autophagy-inducing drugs, including B-cell CLL/lymphoma 2 (BCL2) inhibitors.