No abstract

Purpose: Gene expression profiling identified receptor tyrosine kinase ROR1, an embryonic protein involved in organogenesis, as a signature gene in B-cell chronic lymphocytic leukemia (B-CLL). To assess the suitability of ROR1 as a cell surface antigen for targeted therapy of B-CLL, we carried out a comprehensive analysis of ROR1protein expression. Experimental Design: Peripheral blood mononuclear cells, sera, and other adult tissues from B-CLL patients and healthy donors were analyzed qualitatively and quantitatively for ROR1 protein expression by flow cytometry, cell surface biotinylation,Western blotting, and ELISA. Results: ROR1 protein is selectively expressed on the surface of B-CLL cells, whereas normal B cells, other normal blood cells, and normal adult tissues do not express cell surface ROR1. Moreover, cell surface expression of ROR1 is uniform and constitutive, i.e., independent of anatomic niches, independent of biological and clinical heterogeneity of B-CLL, independent of B-cell activation, and found at similar levels in all B-CLL samples tested. The antibody binding capacity of B-CLL cell surface ROR1was determined to be in the range of 10 3 to 10 4 molecules per cell. A portion of B-CLL cell surface ROR1 was actively internalized upon antibody binding. Soluble ROR1protein was detectable in sera of <25% of B-CLL patients and a similar fraction of healthy donors at concentrations below 200 ng/mL. Conclusions: The restricted, uniform, and constitutive cell surface expression of ROR1protein in B-CLL provides a strong incentive for the development of targeted therapeutics such as monoclonal antibodies.

COVID-19 is a clinical syndrome ranging from mild symptoms to severe pneumonia that often leads to respiratory failure, need for mechanical ventilation, and death. Most of the lung damage is driven by a surge in inflammatory cytokines [interleukin-6, interferon-γ, and granulocyte-monocyte stimulating factor (GM-CSF)]. Blunting this hyperinflammation with immunomodulation may lead to clinical improvement. GM-CSF is produced by many cells, including macrophages and T-cells. GM-CSF-derived signals are involved in differentiation of macrophages, including alveolar macrophages (AMs). In animal models of respiratory infections, the intranasal administration of GM-CSF increased the proliferation of AMs and improved outcomes. Increased levels of GM-CSF have been recently described in patients with COVID-19 compared to healthy controls. While GM-CSF might be beneficial in some circumstances as an appropriate response, in this case the inflammatory response is maladaptive by virtue of being later and disproportionate. The inhibition of GM-CSF signaling may be beneficial in improving the hyperinflammation-related lung damage in the most severe cases of COVID-19. This blockade can be achieved through antagonism of the GM-CSF receptor or the direct binding of circulating GM-CSF. Initial findings from patients with COVID-19 treated with a single intravenous dose of mavrilimumab, a monoclonal antibody binding GM-CSF receptor α, showed oxygenation improvement and shorter hospitalization. Prospective, randomized, placebo-controlled trials are ongoing. Anti-GM-CSF monoclonal antibodies, TJ003234 and gimsilumab, will be tested in clinical trials in patients with COVID-19, while lenzilumab received FDA approval for compassionate use. These trials will help inform whether blunting the inflammatory signaling provided by the GM-CSF axis in COVID-19 is beneficial.

Survival of chronic lymphocytic leukemia (CLL) cells in vivo is supported by the tissue microenvironment, which includes components of the extracellular matrix. Interactions between tumor cells and the extracellular matrix are in part mediated by CD44, whose principle ligand in this respect is hyaluronic acid. Purpose: to evaluate the effect of CD44 engagement on the survival of CLL cells. Experimental Design: CD44 in CLL cells was engaged by anti-CD44 monoclonal antibody, or hyaluronic acid, and the effects of CD44 activation on CLL cell viability and pro-survival pathways were evaluated. Results: engagement of CD44 activated the PI3K/AKT and MAPK/ERK pathways and increased MCL-1 protein expression. Consistent with the induction of these anti-apoptotic mechanisms, CD44 protected CLL cells from spontaneous and fludarabine-induced apoptosis. Leukemic cells of the more aggressive CLL subtype that express unmutated IgVH genes (U-CLL) showed higher CD44 expression than IgVH-mutated CLL (M-CLL) cells, and acquired a greater survival advantage via CD44 activation. Thus, CD44 activation in the tissue microenvironment may contribute to increased MCL-1 protein levels, resistance to apoptosis, and could contribute to the more progressive nature of U-CLL. Furthermore, PI3K or MEK inhibitors as well as obatoclax, an antagonist of MCL-1, blocked the pro-survival effect of CD44. In addition, obatoclax synergized with fludarabine to induce apoptosis of CLL cells. Conclusions: components of the extracellular matrix may provide survival signals to CLL cells through engagement of CD44. Inhibition of MCL-1, PI3K, and MAPK/ERK pathways are promising strategies to reduce the anti-apoptotic effect of the microenvironment on CLL cells.