[Show abstract][Hide abstract] ABSTRACT: Background Proteins in the BCL-2 family are key regulators of apoptosis, or programmed cell death. Navitoclax, a selective inhibitor of both BCL-2 and BCL-X(L) demonstrated efficacy in the IFN-α induced lupus model in NZB/WF1 mice suggesting that dysregulation of this pathway is operative in lupus disease. However, thrombocytopenia caused by BCL-X(L) inhibition limits the chronic use of this therapeutic agent. We have conducted studies to evaluate a highly potent and orally available BCL-2-selective inhibitor, Venetoclax (ABT-199), for efficacy and mechanism of action in lupus prone NZB/WF1 mouse model.
Objectives To determine the efficacy of Venetoclax and the effect on immune cell subsets in lupus prone NZB/WF1 mice.
Methods NZB/WF1 mice were treated daily for 24 weeks with vehicle or Venetoclax. Proteinuria and survival data are presented as Kaplan-Meyer survival curves. Changes in blood lymphocytes and platelets were assessed by Celldyn blood analyzer. IgG deposition and changes in leukocyte subsets in the kidney were evaluated by immunohistochemistry. Circulating anti-dsDNA antibody levels were determined using a semi-quantitative ELISA assay. In a separate study NZB/WF1 mice were treated daily with vehicle or Venetoclax at 30mg/kg for 16 weeks, leukocyte subsets in spleen, kidney and bone marrow were analyzed by flow cytometry
Results Venetoclax dose-dependently reduced the incidence of severe proteinuria compared to vehicle control and conferred 100% survival at the higher doses in NZB/W F1 lupus mice. Venetoclax attenuated glomerulonephritis, tubular dilatation and IgG deposition in the kidney as well as reductions in numbers of B220+, CD3+, F4/80+ and CD138+ cells compared with vehicle-treated mice. Consistent with its BCL-2 selectivity profile, Venetoclax efficacy in NZB/WF1 mice correlated with a dose-dependent reduction of lymphocytes in peripheral blood (45% reduction for ABT-199 11mg/kg vs. vehicle; 70% reduction for ABT-199 100 mg/kg vs. vehicle) with no effect on platelet numbers. Venetoclax also demonstrated a significant reduction in the numbers of splenic T cells (CD4+, CD8+), B cells (transitional 2, germinal center, and mature). Interestingly, other B cell subsets (transitional 1, marginal zone, and B1) were largely unaltered. Venetoclax did not impair early B cell development or the number of CD138+ long-lived plasma cells in the bone marrow. These data were consistent with the unaltered anti-dsDNA titers in these animals.
Conclusions Treatment of lupus prone NZBW/F1 mice with Venetoclax resulted in preservation of renal function and complete protection against severe proteinuria and mortality. Venetoclax treatment resulted in lymphopenia and a reduction of cell infiltration into the kidney while sparing circulating platelets. Splenic marginal zone B cells, the first line of defense against blood-borne pathogens, were resistant to Venetoclax. Taken together, these data underscore the essential role of BCL-2 in the pathogenesis of lupus and support a role for BCL-2 selective inhibition in the treatment of autoimmunity.
Disclosure of Interest L. C. Wang Shareholder of: Abbvie, S. Perper Shareholder of: Abbvie, A. Schwartz Shareholder of: Abbvie, C. Goess Shareholder of: Abbvie, L. O'Connor Shareholder of: Abbvie, D. Hartman Shareholder of: Abbvie, C. Graff Shareholder of: Abbvie, A. Souers Shareholder of: Abbvie, J. Leverson Shareholder of: Abbvie, S. Elmore Shareholder of: Abbvie, L. Olson Shareholder of: Abbvie
No preview · Article · Jun 2015 · Annals of the Rheumatic Diseases
[Show abstract][Hide abstract] ABSTRACT: Background Lupus nephritis (LuN) is the most common, severe manifestation of systemic lupus erythematosus (SLE). We have previously shown that glomerulonephritis appears to be a manifestation of systemic autoimmunity while tubulointerstitial inflammation (TII) is associated with additional, in situ adaptive immune cell networks that might amplify local inflammation and tissue damage. Furthermore, patients with significant TII are more likely to fail conventional therapy and progress to renal failure. Therefore, understanding the mechanisms of in situ adaptive immunity in lupus TII might provide new therapeutic opportunities for treating LuN. In mice, BCL-2 family proteins are key regulators of immune cell apoptosis and forced expression of BCL-2 in B cells promotes a lupus-like nephritis. We hypothesized that dysregulated expression of the BCL-2 family proteins in human LuN TII might contribute to local adaptive immunity, inflammation and disease pathogenesis.
Objectives To determine the expression of BCL2 family proteins in human LuN TII.
Methods Frozen renal biopsy specimens from 26 patients with SLE (ISN/RPS Class III, IV or V LuN), 10 mixed cellular renal allograft rejection (MR) biopsy specimens and tonsil samples from healthy subjects were stained with antibodies specific for CD20, CD4, BCL-2, MCL-1 or BIM. Images were acquired using scanning laser confocal microscopy. IFNa-induced NZB/W F1 mice were treated daily with vehicle or 30 mg/kg of Venetoclax (ABT-199), a BCL-2 selective inhibitor, and analyzed for survival and the development of proteinuria.
Results A recently developed imaging tool (Science Translational Medicine 2014, 6:230) was used to quantify the frequency of CD20+ B and CD4+ T cells expressing the anti-apoptotic molecules BCL-2, MCL-1 and the pro-apoptotic molecule BIM. In primary follicles of normal tonsils, both B and T cells frequently expressed BCL-2. However, upon entry into germinal centers (GC), BCL-2 was down-regulated and MCL-1 was induced. In marked contrast, in LuN and MR TII biopsies, the frequency of BCL-2+ cells was increased in B and T cells while MCL-1+ cells were rare. Observed differences between tonsil GC lymphocytes versus TII in LuN and MR for both BCL-2 and MCL-1 were highly significant (p<0.001). These expression differences were confirmed by laser capture microscopy coupled to qPCR. In contrast, the frequency of BIM+ cells did not significantly vary among tissues. Consistent with these findings, BCL-2, but not MCL-1 expressing cells were detected in the inflamed kidney in IFNa-induced NZB/W F1 lupus mice. Furthermore, administration of Venetoclax, prevented the development of lupus nephritis in these animals.
Conclusions Frequent BCL-2 expressing cells were present in LuN, MR TII tissues and IFNa-induced NZB/W F1 lupus mice. Treatment of these mice with Venetoclax resulted in the preservation of renal function. These data indicate that BCL-2, which is dysregulated in TII, is an attractive therapeutic target in cases of lupus nephritis manifesting tubulointerstitial inflammation.
Disclosure of Interest L. C. Wang Shareholder of: Abbvie Inc., K. Ko: None declared, D. Yanez: None declared, N. Kaverina: None declared, V. Liarski: None declared, Y. Peng: None declared, L. Lan: None declared, S. Perper Shareholder of: Abbvie Inc., A. Schwartz Shareholder of: Abbvie Inc., L. O'Connor Shareholder of: Abbvie Inc., A. Souers Shareholder of: Abbvie Inc., S. Elmore Shareholder of: Abbvie Inc., L. Olson Shareholder of: Abbvie Inc., M. Giger: None declared, M. Clark Grant/research support from: Abbvie Inc.
No preview · Article · Jun 2015 · Annals of the Rheumatic Diseases
[Show abstract][Hide abstract] ABSTRACT: The anti-apoptotic protein MCL-1 is a key regulator of cancer cell survival and a known resistance factor for small-molecule BCL-2 family inhibitors such as ABT-263 (navitoclax), making it an attractive therapeutic target. However, directly inhibiting this target requires the disruption of high-affinity protein-protein interactions, and therefore designing small molecules potent enough to inhibit MCL-1 in cells has proven extremely challenging. Here, we describe a series of indole-2-carboxylic acids, exemplified by the compound A-1210477, that bind to MCL-1 selectively and with sufficient affinity to disrupt MCL-1-BIM complexes in living cells. A-1210477 induces the hallmarks of intrinsic apoptosis and demonstrates single agent killing of multiple myeloma and non-small cell lung cancer cell lines demonstrated to be MCL-1 dependent by BH3 profiling or siRNA rescue experiments. As predicted, A-1210477 synergizes with the BCL-2/BCL-XL inhibitor navitoclax to kill a variety of cancer cell lines. This work represents the first description of small-molecule MCL-1 inhibitors with sufficient potency to induce clear on-target cellular activity. It also demonstrates the utility of these molecules as chemical tools for dissecting the basic biology of MCL-1 and the promise of small-molecule MCL-1 inhibitors as potential therapeutics for the treatment of cancer.
Full-text · Article · Jan 2015 · Cell Death & Disease
[Show abstract][Hide abstract] ABSTRACT: A-1155463, a highly potent and selective BCL-XL inhibitor, was discovered through nuclear magnetic resonance (NMR) fragment screening and structure-based design. This compound is substantially more potent against BCL-XL-dependent cell lines relative to our recently reported inhibitor, WEHI-539, while possessing none of its inherent pharmaceutical liabilities. A-1155463 caused a mechanism-based and reversible thrombocytopenia in mice and inhibited H146 small cell lung cancer xenograft tumor growth in vivo following multiple doses. A-1155463 thus represents an excellent tool molecule for studying BCL-XL biology as well as a productive lead structure for further optimization.
[Show abstract][Hide abstract] ABSTRACT: Because of the promise of BCL-2 antagonists in combating chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL), interest in additional selective antagonists of antiapoptotic proteins has grown. Beginning with a series of selective, potent BCL-XL antagonists containing an undesirable hydrazone functionality, in silico design and X-ray crystallography were utilized to develop alternative scaffolds that retained the selectivity and potency of the starting compounds.