Despite the emergence of small molecule inhibitors, current treatment strategies for chronic lymphocytic leukemia (CLL) are not curative, and the search for new therapeutic modalities continues. Prosurvival signaling derived from the microenvironment is often mediated via JAK signaling. However, whether JAK inhibitors are useful in CLL therapy has not been studied extensively. JAK inhibitors are valuable therapeutic agents in myelofibrosis and show promising results in graft-versus-host-disease. However, JAK inhibition is associated with an increased infection risk, presumably because of the effect on other immune cells, a feature shared with other kinase inhibitors used for CLL treatment, such as the BTK inhibitor ibrutinib and the PI3Kd inhibitor idelalisib. We compared functional effects of the JAK1/2 inhibitors momelotinib and ruxolitinib, the BTK inhibitors ibrutinib and tirabrutinib, and PI3Kd inhibitor idelalisib on malignant CLL cells but also on healthy human T, B, and NK lymphocytes. We found several interesting differences among the inhibitors, apart from expected and well-known effects. Momelotinib but not ruxolitinib blocked cytokine-induced proliferation of CLL cells. Momelotinib also reduced BCR signaling, in contrast to ruxolitinib, indicating that these JAK inhibitors in fact have a distinct target spectrum. In contrast to tirabrutinib, ibrutinib had inhibitory effects on T cell activation, probably because of ITK inhibition. Remarkably, both BTK inhibitors stimulated IFN-g production in a mixed lymphocyte reaction. Collectively, our results demonstrate that kinase inhibitors directed at identical targets may have differential effects on lymphocyte function. Their unique profile could be strategically employed to balance desired versus unwanted lymphocyte inhibition.

Background:Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous B cell malignancy, with accumulation of clonal CD5+ B cells in blood, lymph nodes (LN) and bone marrow. CLL has become a paradigm for a cancer that depends on signals from the microenvironment. This is evidenced by the clinical success of the Btk inhibitor ibrutinib, which drives CLL cells out of the LN to eventually die by neglect. In peripheral blood, CLL cells are quiescent, non‐cycling, with low metabolic activity. In contrast, in LN CLL cells receive various signals from surrounding cells, crucial for proliferation, and protection from chemotherapeutic drugs. These acquired advantages are possibly linked with altered metabolism, but it is unclear if and how CLL cells change their metabolism in the LN environment.Aims:We aimed to investigate the altered metabolism of CLL cells in the tumor microenvironment, and what signals determine these changes.Methods:Glucose uptake, glycolysis, mitochondrial mass, oxidative phosphorylation and lipid uptake were measured by flow cytometer or Seahorse XF96 analyzer, and 90 metabolites were analyzed by LC mass‐spectrometry.Results:We first established that the metabolic activity profile of circulating recent LN emigrants (CXCR4high/CD5dim PB lymphocytes) resembled that of actual LN residents (obtained by a core biopsy), which showed both higher mitochondrial mass and glucose uptake than paired peripheral blood CLL cells. In vitro stimulation of isolated blood‐derived CLL samples demonstrated that CD40 but not BCR signaling recapitulate these metabolic changes. Next, 10 patient samples were stimulated with/without CD40L for 48 hours and analyzed by mass‐spec for metabolic intermediates. To draw a further parallel with the situation in LN, metabolomics was performed on 13 patients sampled before and after 3 months of ibrutinib treatment. Combined, the data shows that various metabolic pathways are activated in the tumor microenvironment, particularly TCA cycle, pyruvate metabolism, glycolysis, and fatty acid metabolism. Apart from these overall changes, the highest ranking shifts in metabolites point to involvement of amino acids to fuel the TCA cycle. Moreover, aberrant patterns in metabolic changes after ibrutinib were noted in some patients, possibly linked with clinical response.Summary/Conclusion:CD40 signaling leads to increased oxidative phosphorylation and glycolysis in the CLL microenvironment. Opposite changes were observed in ibrutinib treated samples, together providing indirect insight into the metabolism in the CLL LN.

Chronic lymphocytic leukemia (CLL) has become a paradigm for a cancer that depends on signals from the microenvironment. In lymph nodes (LN), CLL cells receive from surrounding cells proliferative and pro-survival signals, which can protect against chemotherapy and also the Bcl-2 inhibitor venetoclax (VEN). To avoid drug resistance, combination of VEN with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib (IBR) is currently explored. IBR has the potency to drive CLL cells out of the LN to eventually die by VEN-induced apoptosis. The activation status in the LN likely affects tumor metabolism, though it is yet unclear how, and whether such putative metabolic changes can be linked to VEN resistance. In this study,we aimed to investigate the altered metabolism of CLL cells in the tumor microenvironment (TME), what signals determine these changes, and how to counteract VEN resistance at the metabolic level. We compared the metabolic status of CLL cells in LN biopsy material and paired peripheral blood (PB) CLL cells. Both higher mitochondrial mass and glucose uptake (flow cytometry) were found in CLL cells derived from LN as compared to PB. To determine which TME signals affect metabolism, we mimicked 3 LN signals: CD40, B cell receptor (BCR) and toll-like receptor (TLR) signaling.Invitrostimulation of PB CLL was followed by mitochondrial mass and glucose uptake (flow cytometry), oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measured on Seahorse XF Analyzer. The datademonstrated that CD40 but surprisingly not BCR or TLR signaling recapitulated the metabolic changes observed in LN cells. Next, two sets of metabolomics were performed for samples both in vitro and in vivo. Firstly, PB samples of 10 patients were stimulated with/without CD40L for 48 hours and analyzed by mass-spec for metabolic intermediates. In parallel, metabolomics was performed on PB samples from 13 patients sampled before and after 3 months of ibrutinib treatment. The data show various metabolic pathways are activated in the TME, particularly citric acid (TCA) cycle, pyruvate metabolism, glycolysis, and fatty acid metabolism. Apart from these general changes, the highest-ranking shifts in metabolites point to involvement of amino acids to fuel the TCA cycle, which in turn drives oxidative phosphorylation (OXPHOS). Overall, CD40 signaling results in increased glycolysis, but more dominantly, increased OXPHOS, while IBR in fact has opposing effects (Figure 1), indicating that TME-driven metabolic alterations are mitigated by IBR treatment. In order to link the altered metabolic state to VEN resistance, PB-derived cells were treated with OXPHOS inhibitors during CD40 stimulation. Remarkably, OXPHOS inhibition by oligomycin (ATP synthase inhibitor) did not affect CLL activation, yet counteracted strongly for VEN resistance. Of several BCL-2 family members induced by CD40 ligation, both anti-apoptotic MCL-1 and BCL-XL were downregulated by oligomycin. These data suggest that OXPHOS inhibition affects CD40 signals to counteract VEN resistance. In conclusion, metabolic changes of CLL in LN are recapitulated by CD40 signal, while IBR treatment shows opposite effects, together providing indirect insight into the LN metabolism. In LN, most of the key metabolic pathways are enhanced, particularly OXPHOS. Finally, we found OXPHOS inhibition reverses CLL VEN resistance. Our findings link CLL metabolism in LN microenvironment to VEN resistance, and may provide novel clues for therapeutic targeting in the treatment of VEN resistance patients. Disclosures Forconi: Roche: Honoraria; Gilead Sciences: Research Funding; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Menarini: Consultancy; Novartis: Honoraria; Janssen-Cilag: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau. van der Windt:Genmab: Employment. Eldering:Celgene: Research Funding; Janssen Pharmaceutical Companies: Research Funding; Roche: Research Funding.

Introduction. BCL-2 family members are crucial determinants for survival in normal and malignant B cells. Venetoclax, the BCL-2 targeting drug, was recently clinically approved for CLL. CLL cells cycle between the lymph node (LN) and peripheral blood, and between those compartments display large changes in BCL-XL and MCL-1, which are not targeted by Venetoclax. However, novel BH3 mimetics specific for BCL-XL and MCL-1 are under preclinical development. Ibrutinib is a clinically successful BTK inhibitor that forces Chronic Lymphocytic Leukemia (CLL) cells out of the lymph node. After prolonged application resistance develops in a fraction of patients, who then show fast disease progression. Thus, both Venetoclax and Ibrutinib have potential drawbacks when applied as single agents. For long-term successful application of new drug combinations, it is crucial to understand BCL-2 member functionality of malignant B cells in relation to their normal counterparts. In parallel, we studied clinical samples of CLL patients under Ibrutinib or Venetoclax treatment for changes in BCL-2 family members. Experimental procedures. FACS sorting of tonsil B subsets, mRNA and protein profiling, co-culture of CLL cells, co-IP of BCL-2 members, cell death assays with BH3 mimetics specific for BCL-2, BCL-XL or MCL-1 (ABT-199, WEHI-539, A-1210477), intracellular FACS for BCL-2, BCL-XL and MCL-1 in CLL (CD19/CD5/CXCR4). Results.We mapped the clearly distinct expression profiles for BCL-2 members in tonsillar naïve, germinal center (GC), plasma (PC) and memory B cells, which translate into different BH3 mimetic sensitivity profiles. In brief, naïve and memory cells rely on BCL-2, GC cells on MCL-1, and PC on BCL-XL. These approaches were extended to primary CLL cells. In a LN model where cells are fully resistant to single agent ABT-199 due to induction of MCL-1, BCL-XL and BFL-1, we find that dual or triple BH3 mimetic combinations restore killing. Moreover, there is differential sensitivity to (combinations of) BH3 mimetics between healthy and malignant B cells, suggesting there is a therapeutic window. Furthermore, intracellular FACS staining for BCL-2, MCL-1, and BCL-XL was performed on patients treated with Ibrutinib or Venetoclax. In untreated patients, recent LN emigrants (CD5hi/CXCR4lo) have higher BCL-XL and MCL-1 expression than 'old' returning cells (CD5lo/CXCR4hi). This distinction collapses under Ibrutinib treatment, demonstrating that Ibrutinib affects pro-survival BCL-2 members in vivo. Interestingly, in patients that develop Ibrutinib resistance the MCL-1 or BCL-XL pre-treatment profile reappears. In contrast, under Venetoclax treatment BCL-2 member levels do not change and can even increase in some patients, underlining the different mode of action of the two drugs. The pattern differed among patients (n=5) and increases were observed for BCL-2, BCL-XL, or MCL-1. Conclusions. BH3 mimetics have not only found their way to the clinic, but can also be used as tools for BCL-2 functionality profiling. Secondly, our ex vivo data underline that combination treatment between (paired) BH3 mimetics and/or Ibrutinib may afford better long-term efficacy in CLL than single agents. Thirdly, BCL-2 members may be useful as biomarkers for response and progression. Disclosures Eldering: Roche: Research Funding; Gilead: Research Funding. Brown:Pharmacyclics: Consultancy; Abbvie: Consultancy; Roche: Consultancy; Genetech: Consultancy. Kater:Celgene: Research Funding.