ALBERT

Description: Pim kinases have been shown to play a key role as downstream effectors of growth factor signaling in hematological malignancies. We have previously described AZD1208, a novel, orally bioavailable, highly selective pan Pim kinase inhibitor, undergoing clinical testing in AML. Specifically, we had demonstrated the anti-proliferative activity of AZD1208 in models of AML and had shown that AZD1208 treatment of AML cell lines results in the dose dependent reduction of pBAD at serine 112 and p4EBP1 at serine 65 as well as pp70S6K at threonine 389 and pS6 at serine 235/236, associated with global effects on protein translation (Keeton et al. Blood 2014). Since AZD1208 is about 10-15 fold more potent against Pim-1 than Pim-2 in both enzymatic and cellular assays, we sought to assess the dependency of each of these markers on Pim-1 and/or Pim-2 for phosphorylation using a Pim-1/3 selective inhibitor (Pim-1/3) that lacks Pim-2 inhibitory activity. In vitro studies demonstrate that while the modulation of pBAD does not require inhibition of Pim-2, the regulation of 4EBP1 as well as p70S6K and S6 are dependent, at least in part, on Pim-2 activity. As these proteins are canonically regulated by mTORC1, these data are consistent with Pim-2 acting upstream of this complex; perhaps through phosphorylation of TSC2 as reported for multiple myeloma cell lines (Lu et al. Blood 2013). These observations are also supported by in vivo data. Analysis of pBAD and p4EBP1 levels in Molm16 xenografts show that maximal inhibition of pBAD (70-80% inhibition) is achieved at AZD1208 plasma concentration of about 1000 ng/ml, consistent with the estimated cellular IC90 of pBAD inhibition in vitro. However, maximal inhibition of p4EBP1 is achieved at concentrations 3-5 fold higher. This observation is consistent with the differential potency of AZD1208 against Pim-1 and Pim-2, and with a requirement for Pim-2 inhibition for maximal p4EBP1 inhibition in this AML model. pBAD and p4EBP1 were selected as the pharmacodynamic endpoints for evaluating clinical target inhibition in patient samples. Bone marrow and peripheral blood samples were collected pre- and post-dose from relapsed, refractory AML patients enrolled in the AZD1208 Phase I dose escalation. Analysis of pBAD inhibition on day 1 of dosing shows at least 60-70% inhibition across all of the dosing cohorts. With clinical exposures on day 1 approaching 1000 ng/ml at the first dose level and exceeding 1000 ng/ml as the dose increases, the extent of pBAD inhibition seen in patients appears to be consistent with Pim-1 inhibition, as seen in preclinical models. Furthermore, and also similar to our preclinical observations, maximal inhibition of p4EBP1 in patients is achieved only at higher exposures. These data strengthen the hypothesis that BAD phosphorylation is primarily dependent on Pim-1, whereas suppression of Pim-2 activity is required for maximal inhibition of p4EBP1 in AML cells. In summary, Pim inhibition in AML cell line models and in patients treated with AZD1208 results in the inhibition of the downstream targets of Pim signaling, pBAD and p4EBP1. Invitro and in vivo, the inhibition of pBAD is consistent with inhibition of Pim-1 while inhibition of p4EBP1 indicates a requirement for Pim-2 inhibition as well. These observations are validated in patients and provide further evidence for the relevance of these biomarkers as a measure of Pim signaling in AML. Disclosures McEachern: AstraZeneca: Employment, Equity Ownership. O'Connor:AstraZeneca: Employment, Equity Ownership. DuPont:AstraZeneca: Employment, Equity Ownership. Gibbons:AstraZeneca: Employment, Equity Ownership. Pablo:AstraZeneca: Employment, Equity Ownership. Vishwanathan:AstraZeneca: Employment, Equity Ownership. McCoon:AstraZeneca: Employment, Equity Ownership. Cortes:AstraZeneca: Research Funding. Neumann:AstraZeneca: Employment, Equity Ownership. Keating:AstraZeneca: Employment, Equity Ownership. Pease:AstraZeneca: Employment, Equity Ownership. Brown:AstraZeneca Pharmaceuticals: Employment, Patents & Royalties. Barrett:AstraZeneca: Employment, Equity Ownership.

Description: DNA damage response (DDR) defects, particularly TP53 or biallelic ATM aberrations, are associated with chemoresistance in chronic lymphocytic leukemia (CLL). Chemoimmunotherapy or B-cell receptor signaling inhibitors alone may not be sufficient to overcome adverse prognosis or provide durable response in TP53 or biallelic ATM inactivated CLL. In particular, genomic instability resulting from impaired DDR facilitates rapid clonal evolution leading to treatment refractoriness or disease relapse. Development of therapeutic approaches specifically targeting DDR defects is therefore necessary for effective long-term control of DDR-defective CLL. We previously demonstrated selective cytotoxicity of the ATR inhibitor AZD6738 towards TP53 or ATM null CLL cells, and validated this in CLL xenograft models for biallelic TP53 or ATM loss. Here, we provide mechanistic insight into the synthetically lethal interactions between ATR pathway inhibition and TP53 or ATM loss in CLL, and offer experimental evidence supporting the use of ATR inhibition in combination with conventional chemotherapies and other targeted therapies in CLL. To determine the mechanism of ATR inhibition in CLL, we first investigated its effect on DDR-intact cycling primary cells. We observed compensatory activation of the ATM/p53 pathway in AZD6738-treated cells in response to hydroxyurea, providing evidence for crosstalk between ATR and ATM/p53 pathways. Next, we explored the cellular consequence of ATR inhibition in CLL cells with DDR loss. To determine the impact of ATR inhibition on DNA replication, we performed DNA fiber analysis, which revealed significantly increased replication fork stalling and firing of replication origins upon AZD6738 treatment in ATM/p53-defective CLL. To investigate the effect of ATR inhibition on DNA damage, we measured γH2AX and 53BP1 foci formation, markers of DNA double-strand breaks. Significant induction of γH2AX and 53BP1 foci was seen in ATM/p53-defective CLL cells upon AZD6738 treatment, suggesting that ATR inhibition exacerbates replication stress in ATM/p53 defective cells by imposing requirement for DDR through the ATM/p53 pathway. On the contrary, 53BP1 bodies, a marker of unreplicated DNA, appeared in ATM/p53 proficient but not in ATM/p53 deficient cells following AZD6738 exposure, indicating that cell cycle arrest in response to replication stress is ATM/p53 dependent. This was corroborated by cell cycle profiling and co-labeling experiments of γH2AX with phosphohistone H3 ser10, a marker of mitosis, showing that ATM/p53-defective CLL cells carrying unrepaired DNA damage continued to cycle. Thus, ATR inhibition results in accumulation of intolerable levels of DNA damage in DDR-defective CLL, leading to cell death by mitotic catastrophe, which we have confirmed both in vitro and in AZD6738-treated murine xenograft models. The benefit of ATR inhibition as a therapeutic strategy for DDR-defective CLL lies in its ability to alter the subclonal landscape in favor of less unstable DDR-proficient subclones, which are less susceptible to clonal evolution, thus reducing the likelihood of disease relapse. We reasoned that this provides a strong rationale for addition of AZD6738 to existing therapeutic agents for the treatment of CLL with DDR defects. We demonstrated synergistic and additive effects of low-dose AZD6738 in ATM/p53-defective CLL cells with DNA damaging agents such as chlorambucil, fludarabine, bendamustine and cyclophosphamide, BCR-signaling inhibitors and the PARP inhibitor olaparib. We validated the AZD6738 plus chlorambucil combination in biallelic TP53 or ATM inactivated primary CLL xenografts, where combined AZD6738/chlorambucil treatment was superior to chlorambucil alone, as evidenced by significantly greater reduction in tumor load and percentage of CLL subclones with del(17p) or del(11q) in animals treated with combination regimen compared to single-agent chlorambucil. Similar investigations evaluating AZD6738 plus ibrutinib versus ibrutinib monotherapy in primary CLL xenografts are currently underway. We conclude that ATR inhibition is a suitable approach for targeting the loss of p53 function in aggressive CLL subclones and should be considered as a valuable addition to DNA damaging agents and current targeted treatments. Disclosures Off Label Use: ATR inhibitor AZD6738 targets TP53-null or ATM-null phenotype inducing synthetic lethality. Brown:AstraZeneca Pharmaceuticals: Employment, Patents & Royalties. Lau:AstraZeneca Pharmaceuticals: Employment.

Description: Endowment payouts have become an increasingly important component of universities' revenues in recent decades. We study how universities respond to financial shocks to endowments and thus shed light on a number of existing models of endowment behavior. Endowments actively reduce payouts relative to their stated payout policies following negative, but not positive, shocks. This asymmetric behavior is consistent with “endowment hoarding,” especially among endowments whose current value is close to the benchmark value at the start of the university president's tenure. We also document the effect of negative endowment shocks on university operations, such as personnel cuts. (JEL G35, I22, I23)