Chronic lymphocytic leukemia (CLL), an incurable malignancy of mature B-lymphocytes involves blood, bone marrow, and secondary lymphoid organs. A role of the tissue microenvironment in the pathogenesis of CLL is hypothesized based on in vitro observations but its contribution in vivo remain ill-defined. To elucidate effects of tumor host interactions in vivo we purified tumor cells from 24 treatment nave patients. Samples were obtained concurrently from blood, bone marrow and/or lymph node and analyzed by gene expression profiling. We identified the lymph node as a key site in CLL pathogenesis. CLL cells in the lymph node showed upregulation of gene signatures indicating B-cell receptor (BCR) and NFkappaB activation. Consistent with antigen dependent BCR signaling and canonical NFkappaB activation, we detected phosphorylation of SYK and IkB-alpha, respectively. Expression of BCR target genes was stronger in clinically more aggressive CLL indicating more effective BCR signaling in this subtype in vivo. Tumor proliferation quantified by expression of E2F and c-MYC target genes and verified by Ki67 staining by flow cytometry was highest in lymph node and correlated with clinical disease progression. To allow modeling of pathogenic tumor-host interactions we have established a mouse model that will be instrumental in testing specific interventions. In a second study we have focused on the role of CD44 a receptor for hyaluronic acid. We showed that CD44 engagement protects CLL cells from spontaneous and fludarabine-induced apoptosis. The anti-apoptotic effect appears to be mediated through activation of the PI3K/Akt and MAPK/ERK pathways and increased MCL-1 protein levels. PI3K or MEK inhibitors as well as obatoclax, an antagonist of MCL-1, blocked the pro-survival effect of CD44. Furthermore, obatoclax sensitized CLL cells to fludarabine resulted in a synergistic drug effect. Our findings emphasize the therapeutic potential of PI3K/AKT or MAPK/ERK inhibitors and obatoclax for combination chemotherapy approaches that overcome the supportive effect of the tissue microenvironment on CLL cell survival and drug resistance. These insights open avenues for novel therapies in CLL, in particular for agents that target the BCR signal transduction. To dissect the pathogenic role of different signaling pathways in vivo, we wished to develop a model system that can reproduce the tumor biology encountered in the human LN. We hypothesized that secondary lymphoid compartments (such as the bone marrow (BM) or spleen) of mice may recapitulate the human LN microenvironment. In order to study the effect of the murine microenvironment on human CLL cells we chose to use the recently established NOD scid gamma null (NSG) - human CLL xenograft model (Bagnara et al., Blood 2011). 1 x 108 CFSE labeled CLL PB mononuclear cells (PBMCs) were injected through the tail vein. Mice were bled weekly and sacrificed between 2 and 6 weeks post xenograft. The PB CLL cell count (evaluated as hCD45+, hCD19+, hCD5+ cells) one week after injection averaged 300 cells/L but then rapidly declined and became virtually undetectable by 4 weeks. In contrast, CLL cells persisted in the spleen and BM. We found that CLL proliferation (measured by the fraction of CFSE low cells) was barely apparent early (weeks 1-2) but increased significantly by weeks 3-4. A similar trend was also observed for T-cells. There was no significant difference in the fraction of CFSE low cells among the three different compartments;however, the number of CLL cells in the spleen was significantly higher than in the PB or BM, suggesting increased homing to the spleen. Preliminary results investigating the activation state of the xenografted CLL cells suggests that the murine spleen microenvironment can adequately recapitulate that of the human LN. These results provide further justification for the use of the NSG - human CLL xenograft model to study both the pathogenic mechanisms that contribute to disease progression in the tissue microenvironment and as a pre-clinical model for drug development and assessment. We then used the established system to study the effect of PCI-32765, a specific inhibitor of Brutons tyrosine kinase (Btk), on CLL cells. PCI-32765 has been shown to be well tolerated in CLL with preliminary clinical trial data showing that >85% of patients remained on therapy at a median follow-up of four months. In addition, a significant shrinkage of lymph nodes has been observed in the majority of patients displaying lymphadenopathy. As with other B-cell receptor directed therapies, PCI-32765 results in an initial increase in the absolute lymphocyte count. These observations are not explainable by the available in vitro data, demonstrating the need for in vivo investigation. Mice received PCI-32765 or vehicle in their drinking water at 0.16 mg/ml and were sacrificed between 3 and 4 weeks post xenografting with CLL cells. We found that PCI-32765 treatment resulted in a significant reduction in tumor proliferation compared to mice that received no drug. We also found that activation of CLL cells in the microenvironment could be blocked by PCI-32765. These results demonstrate that targeting Btk is sufficient to block key interactions between tumor cells and the microenvironment and thus warrants the use of PCI-32765 as a targeted agent in CLL. We are currently in the latest stage of preparing a clinical trial using this agent in patients with CLL. We are also investigating the potential of ON 01910.Na (Onconova, Therapeutics) a novel non-ATP competitive compound that can inhibit the PI-3K/Akt/mTOR pathway. We found that ON 01910.Na induced apoptosis of the leukemic cells in all CLL samples tested, without affecting T-cell viability. ON 01910.Na was equal cytotoxic against CLL samples showing adverse biologic and cytogenetic features. To further delineate the biological processes underlying ON 01910.Na induced apoptosis, we performed Gene Expression Profiling (GEP) in CLL cells treated in vitro These studies identified ON 01910.Na as a promising agent in the treatment of CLL with an interesting dual mechanism of action: activation of apoptotic stress signals leading to Noxa and BIM up-regulation, combined with inhibition of the BCR/PI3K/AKT pathway that can block microenvironment-induced survival and proliferation signals. Based on these data we initiated a phase I clinical trial of ON 01910.Na that is ongoing and has reached the highest dosing cohort. We initiated a screen for additional compounds targeting CLL in collaboration with the NHGRI chemical genomics facility. In a first round we screened FDA approved drugs against primary tumor samples from patients. In this screen we identified 5 compounds highly active against CLL cells but not or less toxic to normal lymphocytes. We are extending this screen to additional compounds and collaborate in the pre-clinical and clinical evaluation of one of the lead compounds identified in the first screen.

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Gunti, Sreenivasulu; Herman, Sarah E M; Gottumukkala, Raju V S R K et al. (2018) Polyreactive antibodies in CLL correlate with the level of immunoglobulins not the number of B lymphocytes. Leuk Lymphoma :1-4
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