Search

Supplementary Figures S1-S10

Supplementary Table 1

The antiapoptotic protein BCL2 plays critical roles in regulating lymphocyte development and immune responses, and has also been implicated in tumorigenesis and tumor survival. However, it is unknown whether BCL2 is critical for antitumor immune responses. We evaluated whether venetoclax, a selective small-molecule inhibitor of BCL2, would influence the antitumor activity of immune checkpoint inhibitors (ICI). We demonstrate in mouse syngeneic tumor models that venetoclax can augment the antitumor efficacy of ICIs accompanied by the increase of PD-1+ T effector memory cells. Venetoclax did not impair human T-cell function in response to antigen stimuli in vitro and did not antagonize T-cell activation induced by anti–PD-1. Furthermore, we demonstrate that the antiapoptotic family member BCL-XL provides a survival advantage in effector T cells following inhibition of BCL2. Taken together, these data provide evidence that venetoclax should be further explored in combination with ICIs for cancer therapy.Significance:The antiapoptotic oncoprotein BCL2 plays critical roles in tumorigenesis, tumor survival, lymphocyte development, and immune system regulation. Here we demonstrate that venetoclax, the first FDA/European Medicines Agency–approved BCL2 inhibitor, unexpectedly can be combined preclinically with immune checkpoint inhibitors to enhance anticancer immunotherapy, warranting clinical evaluation of these combinations.This article is highlighted in the In This Issue feature, p. 1

The antiapoptotic protein BCL2 plays critical roles in regulating lymphocyte development and immune responses, and has also been implicated in tumorigenesis and tumor survival. However, it is unknown whether BCL2 is critical for antitumor immune responses. We evaluated whether venetoclax, a selective small-molecule inhibitor of BCL2, would influence the antitumor activity of immune checkpoint inhibitors (ICI). We demonstrate in mouse syngeneic tumor models that venetoclax can augment the antitumor efficacy of ICIs accompanied by the increase of PD-1+ T effector memory cells. Venetoclax did not impair human T-cell function in response to antigen stimuli in vitro and did not antagonize T-cell activation induced by anti–PD-1. Furthermore, we demonstrate that the antiapoptotic family member BCL-XL provides a survival advantage in effector T cells following inhibition of BCL2. Taken together, these data provide evidence that venetoclax should be further explored in combination with ICIs for cancer therapy. Significance: The antiapoptotic oncoprotein BCL2 plays critical roles in tumorigenesis, tumor survival, lymphocyte development, and immune system regulation. Here we demonstrate that venetoclax, the first FDA/European Medicines Agency–approved BCL2 inhibitor, unexpectedly can be combined preclinically with immune checkpoint inhibitors to enhance anticancer immunotherapy, warranting clinical evaluation of these combinations. This article is highlighted in the In This Issue feature, p. 1

Background Acute myeloid leukemia (AML) is a highly heterogeneous disease with multiple prognostic indicators of outcomes to conventional treatments; including age, cytogenetic risk, and presence of genomic abnormalities (NPM1, TP53, FLT3 and IDH1/2). Additionally, overexpression of BCL-2 is a predictor for poor response to chemotherapy and can lead to therapeutic resistance in AML (Campos et. al. Blood, 1993). Venetoclax (Ven), an oral selective BCL-2 inhibitor, in combination with hypomethylating agents (HMA) or low dose cytarabine (LDAC), was recently approved for treatment naive patients (pts) with AML who were not fit for standard induction therapy on the basis of two phase 1b/2 studies (DiNardo et.al, Lancet Onclogy, 2018; Wei et.al., J Clin. Oncol., 2019). Here, we present clinical outcome results from the phase 1b/2 study populations in subgroups defined by molecular markers and correlations with BCL-2 family expression. Methods: The data cut-off dates were 31-Aug-2018 (VEN+HMA study M14-358, NCT02203773) and 22-Aug-2018 (VEN+LDAC study M14-387 NCT02287233). Pt responses were assessed according to the IWG criteria for AML (Cheson et.al., J Clin. Oncol.,2003). The presence of AML associated mutations (NPM1, IDH1, IDH2, TP53 and FLT3) were determined centrally in bone marrow aspirates (BMA) at baseline. BCL-2 (BCL2) gene expression was defined centrally by quantitative polymerase chain reaction (qPCR) using the deltaCt (ΔCt) method. BCL2 expression normalized to a reference gene (2-ΔCt) was evaluated for pts with > 50% AML blasts in baseline BMA (median 75%; range 50-99%). The composite complete remission (CR) and CR with incomplete marrow recovery (CRi) rate, overall survival (OS), time to first response (TTR), and duration of response (DOR), and BCL-2 gene expression were correlated with presence of baseline molecular markers. Results: Data from 209 pts who received Ven 400 mg or 600 mg in combination with either HMA or LDAC are reported here. The median age was 74 years (range: 61-90 years) and 61% (n=127) were male (Table 1). Of the 167 pts with centrally assessed molecular markers, IDH1 or IDH2 mutations were detected in 25.7%, NPM1 mutations in 15.6%, TP53 mutations in 22.2%, and FLT3 mutations in 18.0% pts. Pts may have more than one of these mutations (i.e co-mutations of NPM1 and IDH1or IDH2, NPM1 with FLT3, or NPM1 with TP53) identified in baseline BMA. The CR/CRi rates were 83.7% for pts with IDH1/IDH2 mutations, 84.6% for pts with NPM1 mutations, 59.5% for pts with TP53 mutations, and 53.3% for pts with FLT3 mutations (Table 2). The median OS was not reached (NR) for pts with IDH1, IDH2 or NPM1 mutations, while the median OS for pts with detectable TP53 mutations or FLT3 mutations was 8.9 mos and 12.4 mos respectively. The remissions (CR or CRi) were rapid (median TTR between 1.1 mos to 1.8 mos) for each group (Table 2) and durable responses were observed for pts with IDH1, IDH2, NPM1, or FLT3 mutations (median DOR was either NR for IDH and NPM1 and 19.9 mos for FLT3). Pts with TP53 mutations are known to have poor prognosis and the DOR in this cohort was 5.6 mos. The median time on study was 11.6 mos (range: 0.3-44 mos). The range of BCL2 mRNA expression (2-ΔCt) in bone marrow blasts was 0.03 to 2.93, with a median of 0.86. The Cox hazard ratio for OS was 1.06, p=0.8565 based on BCL2 above the median (> 0.86 2-ΔCt). The multivariate analysis included age, cytogenetic risk, AML type (primary vs secondary) and trial (M14-387 vs M14-358). Albeit not statistically significant and small sample sets, pts with either IDH1 or IDH2 mutations (N = 21) tended to have higher BCL2 expression (median 2-ΔCt 1.1 ; range 0.19 -2.93) while pts with TP53 mutations (N = 7) had lower levels of BCL2 expression (median 2-ΔCt 0.55; range 0.03 - 1.27). Determination of baseline expression of other family members (BCL2L1, MCL1, BCL2A1, BCLw, NOXA, etc) is ongoing and additional univariate and multivariate analyses will be presented at the meeting. Conclusions: These analyses demonstrate that VEN + HMA or LDAC has efficacy across multiple molecular markers in AML. This activity is rapid and durable, and is observed across different levels of BCL2 expression in AML blasts. Disclosures Chyla: Abbvie, Inc: Employment, Other: Stock or options. Harb:AbbVie Inc: Employment, Other: Stock/stock options. Mantis:AbbVie Inc: Employment, Other: Stock/stock options. Riehm:AbbVie Inc.: Employment, Other: Stock/stock options. Ross:AbbVie: Employment, Other: Stock/stock options. Sun:AbbVie: Employment, Other: Stock/stock options. Huang:AbbVie Inc: Employment, Other: Stock/stock options. Jiang:AbbVie Inc.: Employment, Other: Stock/stock options. Dail:Genentech: Employment, Equity Ownership. Peale:Genentech Inc.: Employment, Other: Stock/stock options. Potluri:AbbVie, Inc.: Employment, Other: Stock/stock options. Hayslip:AbbVie Inc: Employment, Other: Stock/stock options. OffLabel Disclosure: Venetoclax is a BCL-2 inhibitor that is FDA-approved in some indications. This presentation will focus on venetoclax for treatment in acute myeloid leukemia, which is not an approved indication.

Little consideration has been given to the possibility that there could be variations in protein expression that alter susceptibility to tumorigenesis without causing other obvious phenotypic effects. Therefore, we sought to determine if haploinsufficiency for the anti-apoptotic protein Bcl-x(L) would affect tumorigenesis. We chose to study Bcl-x(L) because although bcl-x+/- mice were thought to be phenotypically normal, we and others found that haploinsufficiency for Bcl-x(L) lowers fibroblast resistance to apoptosis in tissue culture. Since resistance to certain forms of apoptosis is required for tumor formation, this suggested that decreased Bcl-x(L) expression would afford protection against tumorigenesis. Indeed, we demonstrate here that bcl-x+/- mice are strikingly resistant to carcinogen-induced tumorigenesis. However, we found that they pay a price for their resistance in that they are more susceptible to clinically relevant forms of tissue injury--they suffer increased hepatic injury in a model of binge alcohol abuse and in response to TNF-alpha treatment. These findings are important because they suggest that even minor variations in Bcl-x(L) expression could affect susceptibility to cancer and other diseases. Additionally, they indicate that the potential for increased susceptibility to tissue injury must be considered in the design of chemopreventative and antineoplastic strategies that involve inhibition of Bcl-x(L) activity.

Deamidation of two asparagines activates a conditional PEST sequence to target Bcl-xL for degradation., The cellular concentration of Bcl-xL is among the most important determinants of treatment response and overall prognosis in a broad range of tumors as well as an important determinant of the cellular response to several forms of tissue injury. We and others have previously shown that human Bcl-xL undergoes deamidation at two asparaginyl residues and that DNA-damaging antineoplastic agents as well as other stimuli can increase the rate of deamidation. Deamidation results in the replacement of asparginyl residues with aspartyl or isoaspartyl residues. Thus deamidation, like phosphorylation, introduces a negative charge into proteins. Here we show that the level of human Bcl-xL is constantly modulated by deamidation because deamidation, like phosphorylation in other proteins, activates a conditional PEST sequence to target Bcl-xL for degradation. Additionally, we show that degradation of deamidated Bcl-xL is mediated at least in part by calpain. Notably, we present sequence and biochemical data that suggest that deamidation has been conserved from the simplest extant metazoans through the human form of Bcl-xL, underscoring its importance in Bcl-xL regulation. Our findings strongly suggest that deamidation-regulated Bcl-xL degradation is an important component of the cellular rheostat that determines susceptibility to DNA-damaging agents and other death stimuli., Author Summary Cellular levels of the pro-survival protein Bcl-xL are an important determinant of cellular susceptibility to many death stimuli, including most cancer therapies. We previously showed that human Bcl-xL undergoes deamidation – the conversion of two neutral asparaginyl side-chains into negatively charged aspartyl side-chains – a process that occurs spontaneously but is accelerated by the treatment of tumor cells with DNA-damaging agents. Here, we show that deamidation activates a hitherto undetected signal sequence within Bcl-xL that targets it for degradation by a pathway involving the proteolytic enzyme calpain. This increased degradation of Bcl-xL, and the consequent enhanced cellular susceptibility to programmed cell death, may contribute to the ability of DNA-damaging agents to kill tumors. We also demonstrate that deamidation of Bcl-xL has likely been conserved from the simplest metazoans to humans, underscoring the importance of deamidation in the regulation of Bcl-xL.

The proliferation of all nontransformed adherent cells is dependent upon the development of mechanical tension within the cell; however, little is known about the mechanisms by which signals regulated by mechanical tension are integrated with those regulated by growth factors. We show here that Skp2, a component of a ubiquitin ligase complex that mediates the degradation of several proteins that inhibit proliferation, is upregulated when increased mechanical tension develops in intact smooth muscle and that its upregulation is critical for the smooth muscle proliferative response to increased mechanical tension. Notably, whereas growth factors regulate Skp2 at the level of protein stability, we found that mechanical tension regulates Skp2 at the transcriptional level. Importantly, we demonstrate that the calcium-regulated transcription factor NFATc1 is a critical mediator of the effect of increased mechanical tension on Skp2 transcription. These findings identify Skp2 as a node at which signals from mechanical tension and growth factors are integrated to regulate proliferation, and they define calcium-NFAT-Skp2 signaling as a critical pathway in the mechanoregulation of proliferation.