ALBERT

Description: The evasion of apoptosis, or programmed cell death, is a hallmark of cancer, which promotes tumor initiation and progression. The evasion is in part attributable to the over-expression of anti-apoptotic proteins in the Bcl-2 family. In addition, chemotherapy and radiation can upregulate the expression of the Bcl-2 family in cancer cells, which renders them more resistance to cancer therapy. The most common Bcl-2 family member over-expressed in many solid tumor cells and a fraction of leukemia and lymphoma cells is Bcl-XL and its expression is also highly correlated with resistance to cancer therapy independent of p53 status in many cancers. Therefore, Bcl-XL is one of the most important validated cancer cell targets. Inhibition of Bcl-XL with a small molecule inhibitor has been extensively exploited as a molecularly targeted therapeutic strategy against cancer, resulting in the discovery of several Bcl-2/XL and Bcl-XL inhibitors as promising anti-cancer drug candidates including navitoclax. Unfortunately, these inhibitors failed to become anticancer drugs because platelets are also dependent on Bcl-XL for survival. Therefore, inhibition of Bcl-XL with Bcl-2/XL and Bcl-XL inhibitors causes severe reduction in platelets or thrombocytopenia, an on-target and dose-limiting toxicity, which prevents their use as an effective anticancer drug in clinic. To overcome this problem, we generated a series of novel bifunctional molecules that targeting Bcl-XL to the ubiquitin-proteasome system (UPS) for degradation. These synthetic proteolytic compounds, termed synthetic proteolytics (Syntholytics) or proteolysis targeting chimeras (PROTACs), were rationally designed to recruit the Von Hippel Lindau (VHL) E3 ligase to ubiquitinate Bcl-XL for degradation by the proteasome. Because VHL is minimally expressed in platelets, our Bcl-XL Syntholytics can selectively induce Bcl-XL degradation in various cancer cells but not in platelets. Amongst these Bcl-XL Syntholytics, DT2216 was found to be the most potent in inducing Bcl-XL degradation leading to the loss of viability of Bcl-XL-dependent T-ALL MOLT-4 cells at nanomolar concentrations but did not cause any platelet toxicity. Compared to navitoclax, DT2216 is more potent in induction of apoptosis in a variety of cancer and leukemia cells in vitro in a caspase-dependent manner. Furthermore, our in vivo studies in immunocompromised mice revealed that DT2216 at 15 mg/kg/wk potently inhibited tumor growth in Bcl-XL-dependent MOLT-4 T-ALL xenografts as a single agent whereas navitoclax had no significant effect at the same dosage. Dosing with DT2216 at 15 mg/kg every four days significantly regressed larger established MOLT-4 T-ALL tumors that failed to respond to navitoclax treatment. To assess the therapeutic potential of DT2216 in combination with other Bcl-2 family inhibitors, we employed the Bcl-2/xl dependent NCI-H146 small cell lung cancer cells and the Mcl1/Bcl-xl dependent multiple myeloma EJM cells. The combination of DT2216 with Bcl-2 inhibitor (ABT199) or Mcl-1 inhibitor (S63845) synergistically reduced the viability of H146 and EJM cells, respectively. DT2216 in combination with ABT199 effectively inhibited tumor growth in H146 xenografts. Collectively, our findings suggest that targeting Bcl-XL using Bcl-XL Syntholytics can selectively kill Bcl-XL-dependent T-ALL cells and various solid tumor cells without causing significant platelet toxicity. Moreover, the combination of Bcl-XL Syntholytics with other Bcl-2 protein inhibitors could be used to effectively target multiple cancer types including both hematological and solid tumors. Therefore, Bcl-XL Syntholytics have the potential to be developed as safer and more potent novel anti-cancer drugs. Keywords: Bcl-XL, VHL, Protein degradation, T-ALL, Cancer, Apoptosis Disclosures: S.K., X.Z., D.L., Y.H., P.Z., X. L., G. Z., and D.Z. are inventors of a pending patent application for use of Bcl-xl syntholytics as anti-cancer agents. R.H, G.Z. and D.Z. are co-founders of Dialectic Therapeutics that develops Bcl-xl syntholytics. Disclosures Khan: Dialectic Therapeutics: Patents & Royalties. Lv:Dialectic Therapeutics: Patents & Royalties. He:Dialectic Therapeutics: Patents & Royalties. Zhang:Dialectic Therapeutics: Patents & Royalties. Liu:Dialectic Therapeutics: Patents & Royalties. Konopleva:Stemline Therapeutics: Research Funding. Zheng:Dialectic Therapeutics: Consultancy, Equity Ownership, Patents & Royalties.

Description: Senescent cells (SnCs) accumulate with age. Genetic or pharmacological clearance of SnCs delays several age-associated disorders and extends the healthspan of both progeroid and wild-type (WT) mice (Baker DJ et al. Nature, 479: 232, 2011; Baker DJ et al. Nature, 530: 184, 2016). In addition, our previous study showed that transient depletion of SnCs could rejuvenated aged hematopoietic stem cells (HSCs) and muscle stem cells in normally aged mice (Chang J et al. Nat Med, 22:78, 2016). These findings suggest that SnCs play a causative role in aging and certain aging-related pathologies, and that selective clearance of SnCs with a senolytic agent that can selectively kill SnCs can be beneficial in part by rejuvenating aged tissue stem cells. Thus, senolytic drugs are potential novel anti-aging agents. However, SnCs can re-accumulate within a few weeks after depletion in mice, suggesting that long-term and intermittent clearance of SnCs is required to extend healthspan, as shown in previous studies (Baker DJ et al. Nature, 479: 232, 2011; Baker DJ et al. Nature, 530: 184, 2016). It is not known how long-term and intermittent clearance of SnCs affects tissue stem cells such as HSCs because HSCs are mostly quiescent in order to self-renew and prevent exhaustion. We used p16-3MR transgenic mice to evaluate the effect on HSCs of long-term and intermittent clearance of SnCs by ganciclovir (GCV), which selectively kills SnCs in these mice. We treated WT and p16-3MR mice with GCV (25 mg/kg/day x 5 days/per cycle with a 2-week interval between the cycles by ip injection) or saline (vehicle control) from 12 to 24 months of age. GCV effectively prevented SnC accumulation in p16-3MR mice but had no effect in WT mice. In general, aged p16-3MR mice treated with GCV looked healthier than old vehicle-treated p16-3MR mice and old WT mice treated with vehicle or GCV as expected (Baker DJ et al. Nature, 530: 184, 2016). Importantly, GCV abrogated aging-induced increases in p16 expression, p38MAPK activation, and accumulation of DNA double-strand breaks in HSCs in p16-3MR mice. This abrogation was associated with a significant improvement in HSC clonogenicity, suggesting that long-term and intermittent clearance of SnCs prevents HSC aging in naturally aged mice. This suggestion is confirmed by competitive serial and limited dilution HSC transplantation assays, which revealed that HSCs from GCV-treated old p16-3MR mice were functionally indistinguishable from those of young mice. To elucidate the mechanism by which SnC clearance prevents HSC aging, we evaluated the clonogenicity of Lin−Sca1+c-Kit+ (LSK) cells from young or vehicle- or GCV-treated old p16-3MR mice in co-cultures with bone marrow stromal cells (BMSCs) from these mice. We found that clearance of SnCs not only prevented the decline in HSC clonogenic function with aging but also preserved the hematopoietic supporting function of BMSCs. This preservation was associated with reduced levels of Cdkn2a and Cdkn1a mRNAs and senescence-associated secretory phenotype (SASP) factors (such as Il1β, Tnfα and Ccl5), which can adversely affect HSC self-renewal, and decrease of adipogenic and osteoclastic differentiation. In addition, the levels of the HSC self-renewal promoting factor Spp1 mRNA was higher in BMSCs from GCV-treated old p16-3MR mice than in the cells from vehicle-treated old p16-3MR mice. These findings suggest that long-term and intermittent clearance of SnCs can also inhibit BMSC senescence, which contributes to the prevention of HSC aging. Disclosures He: University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Chang:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Kim:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Almeida:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Campisi:Unity Biotechnology: Equity Ownership, Other: co-founder of Unity Biotechnology which develops small-molecule senolytic drugs. Zhou:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents; Unity Biotechnology: Equity Ownership, Other: Co-founder of Unity Biotechnology which develops small-molecule senolytic drugs; Dialectic Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents.

Description: There is an urgent need for new therapeutic agents to treat patients with T-cell lymphoma (TCL). Multiple hematologic malignancies evade apoptosis through overexpression of anti-apoptotic proteins in the BCL-2 family, including BCL-2, BCL-XL, and MCL-1. We and others recently showed that a large fraction of cutaneous and peripheral TCL cell lines, patient-derived xenografts and primary patient samples depend on BCL-XL for survival (Koch et al, Blood. 2019; 133:566-575). These findings suggest that targeted inhibition of BCL-XL could offer therapeutic benefit for some TCL patients. Currently available small molecule BCL-XL inhibitors have failed during clinical development due to on-target and dose-limiting thrombocytopenia, as platelets depend on BCL-XL for survival. To overcome this toxicity, we developed DT2216, a novel proteolysis-targeting chimera (PROTAC) that targets BCL-XL to the Von Hippel Lindau (VHL) E3 ligase for proteasomal degradation. We selected the VHL ligase because platelets express very low levels of VHL, suggesting that they would be spared from the pro-apoptotic effects of DT2216. Here we examined the therapeutic potential of DT2216 against different TCL cell lines in vitro and in TCL xenograft mouse models. We first profiled the expression of different anti-apoptotic BCL-2 family proteins in multiple TCL cell lines (Fig. 1a) and tested their sensitivity to selective inhibitors for different BCL-2 family proteins as well as commonly-used chemotherapeutic agents (Table 1). The results showed that the TCL cells with higher levels of BCL-XL were more resistant to doxorubicin, etoposide and vincristine. DT2216 was more effective in reducing the viability of BCL-XL-dependent TCLs such as MyLa cells than ABT263 (a dual BCL-2/XL inhibitor). The EC50 value of DT2216 for MyLa TCL cells was less than 10 nM and DT2216 killed MyLa cells through induction of BCL-XL degradation and cellular apoptosis (Fig. 1b-f). Moreover, DT2216 was less toxic to human platelets than ABT263 in vitro with an EC50 〉 3 μM. We further validated the effect and specificity of DT2216 in MJ cells, another BCL-XL-dependent TCL cell line, and demonstrated that its antitumor activity was dependent on proteasome activity. In vivo, when DT2216 was given to mice with MyLa TCL xenografts by i.p. injections at 10 mpk/q4d, it significantly inhibited tumor growth, whereas ABT263 at the same dose had no significant effect (Fig. 1g). More importantly, after MyLa xenografted mice failed to respond to ABT263 treatment, we subjected the mice to DT2216 (10 mpk/q4d), which induced rapid tumor regression and increased the survival of the mice without causing significant reduction of blood platelets (Fig. 1h). These effects were associated with a significant reduction in BCL-XL expression and activation of caspase cascade in tumor xenografts. However, some TCLs depend on not only BCL-XL but also BCL-2 or MCL-1 for evasion of apoptosis. Therefore, we next assessed the therapeutic potential of DT2216 in combination with a selective BCL-2 inhibitor using the TCL PDX DFTL-28776, which depends on both BCL-XL and BCL-2 for survival. We found that the combination of DT2216 with ABT199 (a selective BCL-2 inhibitor) could more effectively kill DFTL-28776 TCL cells than either agent alone in cell culture. The effect of the combination treatment on the growth of DFTL-28776 PDX in vivo is under examination. Collectively, our findings suggest that targeting BCL-XL using DT2216 can selectively kill BCL-XL-dependent TCL cells without causing significant platelet toxicity. Moreover, the combination of DT2216 with an inhibitor targeting other anti-apoptotic BCL-2 family proteins may have broad therapeutic utility against multiple TCL types and other cancers dependent on BCL-XL. Disclosures He: University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Khan:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer agents. Zhang:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer agents. Zheng:Dialectic Therapeutics: Equity Ownership, Other: Co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents; University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Weinstock:Celgene: Research Funding. Zhou:Dialectic Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents; Unity Biotechnology: Equity Ownership, Other: Co-founder of Unity Biotechnology which develops small-molecule senolytic drugs; University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents.

Description: Background The analyses of protein synthesis, accumulation and regulation during grain development in wheat are more complex because of its larger genome size compared to model plants such as Arabidopsis and rice. In this study, grains from two wheat cultivars Jimai 20 and Zhoumai 16 with different gluten quality properties were harvested at five development stages, and were used to displayed variable expression patterns of grain proteins. Results Proteome characterization during grain development in Chinese bread wheat cultivars Jimai 20 and Zhoumai 16 with different quality properties was investigated by 2-DE and tandem MALDI-TOF/TOF-MS. Identification of 117 differentially accumulated protein spots representing 82 unique proteins and five main expression patterns enabled a chronological description of wheat grain formation. Significant proteome expression differences between the two cultivars were found; these included 14 protein spots that accumulated in both cultivars but with different patterns and 27 cultivar-different spots. Among the cultivar-different protein spots, 14 accumulated in higher abundance in Jimai 20 than in Zhoumai 16, and included NAD-dependent isocitrate dehydrogenase, triticin precursor, LMW-s glutenin subunit and replication factor C-like protein. These proteins are likely to be associated with superior gluten quality. In addition, some proteins such as class II chitinase and peroxidase 1 with isoforms in developing grains were shown to be phosphorylated by Pro-Q Diamond staining and phosphorprotein site prediction. Phosphorylation could have important roles in wheat grain development. qRT-PCR analysis demonstrated that transcriptional and translational expression patterns of many genes were significantly different. Conclusions Wheat grain proteins displayed variable expression patterns at different developmental stages and a considerable number of protein spots showed differential accumulation between two cultivars. Differences in seed storage proteins were considered to be related to different quality performance of the flour from these wheat cultivars. Some proteins with isoforms were phosphorylated, and this may reflect their importance in grain development. Our results provide new insights into proteome characterization during grain development in different wheat genotypes.