ALBERT

Description: Altered DNA repair mechanisms are responsible for survival of leukemia stem cells (LSCs) and/or leukemia progenitor cells (LPCs) accumulating numerous lethal DNA double-strand breaks (DSBs). DSBs resulting from stalled/broken replication forks in proliferating cells are primarily repaired by RAD51-mediated homologous recombination repair (HR), which depends on BRCA1-PALB2-BRCA2-RAD51 paralogs (BRCA pathway), while RAD52 pathway serves as redundant back-up mechanism. Enhanced self-renewal of LSCs and high proliferation rate of LPCs commit them to HR. It has been reported that inhibition of RAD52 either by the knockout, specific shRNA, or a small peptide aptamer induced synthetic lethality in BRCA pathway-deficient tumor cell lines and primary leukemia cells. Yet pharmacological inhibition of RAD52, which binds single-stranded DNA (ssDNA) and lacks enzymatic activity, has not been demonstrated. Here, we applied high-throughput screening and structure-based selection followed by biochemical assays and computer modeling to identify three leading compounds: (1) 20264, (2) RU-0084339, and (3) D-I03. Compound 20264 appeared to interact with the hotspot in RAD52 DNA binding domain 1 to interfere with ssDNA binding. RU-0084339 is a major allosteric inhibitor of RAD52 ssDNA binding domain which disassembles undecamer ring structure of RAD52. D-I03 abrogated RAD52-mediated ssDNA annealing and ssDNA pairing. RAD52 small molecule inhibitor (RAD52smi) reduced recruitment of RAD52 to DNA damage-induced nuclear foci and suppressed RAD52-mediated DNA double-strand break (DSB) repair activity in cells with negligible effects on other DSB repair pathways. Importantly, RAD52smi selectively eliminated cancer cell lines carrying BRCA1/2 inactivating mutations. Since inactivating mutations in BRCA pathway are rare in leukemias, individual BRCA pathway-deficient leukemias were identified by Gene Expression and Mutation Analysis (GEMA). Gene Expression approach applied microarrays, qRT-PCR, and/or flow cytometry to identify individual leukemias displaying downregulation of at least one gene in BRCA pathway. On the other hand, Gene Mutation strategy detected individual leukemias expressing an oncogene causing downregulation of BRCA pathway gene(s) (e.g., BCR-ABL1, MLL-AF9, AML1-ETO - mediated downregulation of BRCA1 and/or BRCA2) and harboring inactivating mutations in BRCA pathway (e.g., BRCA2 = FANCD1, and other FA genes). BRCA-deficient cells from individual patients indentified by GEMA were selectively sensitive to RAD52smi alone or in combination with already approved cytotoxic drugs. RAD52 is a promising new target because it is "druggable" by small molecule inhibitors. Moreover, inhibition of RAD52 by genetic knockout and small peptide aptamer did not exert any major negative effects in normal cells and tissues. Altogether, this work provided foundation for precision medicine guided synthetic lethality in BRCA-deficient leukemias exerted by small molecule inhibitors targeting novel mechanism - RAD52 dependent DSB repair. Disclosures No relevant conflicts of interest to declare.

Description: Murine embryonic stem (ES) cells are pluripotent, and may be of potential use for cell replacement and gene therapy. By understanding the mechanism of survival and anti-apoptosis of ES cells, it may be possible to enhance use of ES cells for clinical usage. Our previous studies indicated that undifferentiated ES cells converted into polyploidy instead of apoptosis under the stress. However, differentiated ES cells went through apoptosis in the same manner as mature cells. Oct-4 is a marker for undifferentiated ES cells. We wondered if Oct-4 might be a key player for choosing between polyploidy and apoptosis of ES cells under the stress. To address this question, Oct-4 tetracycline conditional knockout cell line ZHBtc4, received from Dr Austin Smith, was used. To evaluate whether Oct-4 was essential for stress-induced apoptosis of murine ES cells, we used three methods to induce stress: etoposide treatment, heat shock and UV exposure. Four treatment groups were studied, A: Oct-4 knock down ZHBtc4; B: Control ZHBtc4; C: Tetracycline treated CGR8 cell line; D: Control CGR8. CGR8 is the wild type parental cell line for ZHBtc4. These latter two groups were added to eliminate potential effects of tetracycline. After knockdown of Oct-4 for 24 hours, we induced apoptosis by etoposide, heat shock or UV exposure. Apoptosis for Oct-4 knocked down ES cells was significantly increased in response to all stress situations. This suggests that Oct-4 is important for modulating stress-induced apoptosis of ES cells. In order to address potential mechanisms for these effects, we focused on one member of the IAP family—Survivin. Survivin is a well known anti-apoptosis protein. In addition, Oct-4 and survivin are both specifically expressed in embryonic tissue, germ line and tumor tissues (such as breast, pancreatic and colon cancer), but not in other tissues or at low levels in these cells. We hypothesized that the mechanism of Oct-4 related anti-apoptosis may be mediated through Survivin. Towards this possibility, we transfected the Survivin promoter into ES cells after knockdown of Oct-4. Promoter activity of the group with knocked down Oct-4 expression was dramatically decreased, indicating that Oct-4 mediated anti-apoptosis may act through Survivin. Western blots showed that Oct-4 knocked down ES cells had decreased Survivin protein expression. STAT3 is a well known transcription factor. Studies have shown that LIF-induced STAT3 is responsible for ES cell survival. Also, it was reported that STAT3 regulated survivin in breast cancer. We tested whether Oct-4 regulated Survivin through STAT3. Western Blot analysis showed that down regulated Oct-4 induced decreased phosphorylation of STAT3 and expression of total STAT3 protein. This suggests that Oct-4 is essential for anti-apoptosis of ES cells in response to stress, and this effect may be mediated through the STAT3/Survivin pathway.

Description: The signals that mediate T-cell infiltration during T-cell autoimmune diseases are poorly understood. The chemokine CCL21 (originally isolated by us and others as Exodus-2/6Ckine/SLC/TCA4) is highly potent and highly specific for stimulating T-cell migration. However, it is thought to be expressed only in secondary lymphoid organs, directing naive T cells to areas of antigen presentation. It is not thought to play a role in T-cell effector function during a normal immune response. In this study we tested the expression of T-cell chemokines and their receptors during T-cell autoimmune infiltrative skin diseases. By using immunohistology it was found that the expression of CCL21 but not CCL19 or 20 was highly induced in endothelial cells of T-cell autoimmune diseases. The receptor for CCL21, CCR7, was also found to be highly expressed on the infiltrating T cells, most of which expressed the memory CD45Ro phenotype. These data imply that the usual loss of CCL21 responsiveness in the normal development of memory T-cell effector function does not hold for autoimmune skin diseases.

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: Maintenance of a euploid ES cell genome during ex-vivo expansion and differentiation to hematopoietic cells is essential for their safe use in therapeutic intervention and regenerative medicine. It is estimated that about half of all spontaneous mutations that occur in cultured murine ES cells are attributable to chromosomal non-disjunction and chromosome loss. This type of mutation is essentially undetectable in somatic cell cultures of comparable culture age. The mechanisms responsible for this difference between ES and somatic cells are unknown, but are likely related to the absence of a rigorous G1 cell cycle checkpoint and other peculiarities in ES cell cycle regulation compared to somatic cells. The fate of aneuploid ES cells during differentiation has not been systematically studied. Here, we report a remarkable tolerance for aneuploidy/polyploidy in murine ES cells in-vitro when challenged by mitotic stress that is induced by failed cell division followed by mitotic slippage resulting in stable, cycling, tetraploid/polyploid ES cell lines (oscillating between 4N and 8N). We present evidence supporting the idea that canonical apoptosis is uncoupled from mitotic checkpoints in undifferentiated ES cells in contrast to somatic cells and embryoid body (EB) cells. We also present evidence suggesting similar behavior occurs in human ES cell cultures. Uncoupling was associated with low levels of the pro-apoptotic form of phospho-BAD (p-ser128) combined with high expression of anti-apoptotic Survivin in ES compared to EB cells. This suggests uncoupling from mitotic checkpoints may be related to a heightened apoptotic threshold in ES cells compared to EB/somatic cells. Interestingly, culture of ES cells under hypoxic conditions also generated polyploid cells. The polyploid ES cells did not appear to be trophoblastic cells because they continue to express SSEA-1 and other markers of pluripotency. However, we demonstrate that (re-)coupling occurs very early in the differentiation process because only diploid ES cells contribute to EB formation while tetraploid cells do not when EBs are generated from tetraploid/diploid-mosaic cultures. This is, to our knowledge, the first evidence that there may be a potent in-vitro barrier to cells with numerical chromosomal aberrations from contributing to differentiated cells to be used in therapeutic settings. Finally, using a conditional oct-4 knock-down ES cell line, we demonstrate that the self-renewal regulating transcription factor, oct-4, is essential for maintaining the aneuploidy/polyploidy tolerant state. We conclude that aneuploidy/polyploidy-tolerance in pluripotent ES cells in-vitro is an expected occurrence when viewed within the context of ex-vivo ES cell culture where apoptotic culling of cells with chromosomal aberrations, which normally occurs in vertebrate embryos during the peri-implantation period in-vivo, has been artificially interrupted by ES cell derivation. This behavior likely contributes significantly to spontaneous aneuploidization in murine and human ES cell cultures. These data not only lend insight into mechanisms of aneuploidy in ES cell cultures but may also have implications for mechanisms of aneuploidy during tumorigenesis.

Description: Murine embryonic stem (ES) cells may be of potential use for cell replacement and gene therapy. Maintenance of ES cells in an undifferentiated and proliferative state depends on cytokines either secreted by ES cells and/or added to the medium. By understanding the production and release of cytokines in ES cell culture, it may be possible to enhance use of ES cells for clinical usage. Our previous studies indicated that SDF-1/CXCL12, secreted by ES cells, enhances survival, chemotaxis, and hematopoietic differentiation of murine ES cells (Guo et al, Stem cells, in press, 2005). To evaluate whether other cytokines were produced by murine ES cells, we generated conditioned medium (CM) from these cells in the presence of LIF, while the ES cells were in an undifferentiated Oct-4 expressing state, and assayed the CM for cytokines, chemokines, and other growth modulatory factors. ES cell CM enhanced survival in vitro of ES cells subjected to delayed addition of serum to ES cell cultures. Without serum, ES cells didn’t grow in low cell density. However, with CM, ES cells formed colonies at about 63% of the growth of the ES cells in the presence of serum. ES cell CM also enhanced survival of normal murine bone marrow myeloid progenitors (CFU-GM) subjected to delayed growth factor addition in vitro and decreased the rate of apoptosis in murine bone marrow c-kit+lin− cells as assessed by Annexin V assay. Our data showed ES cell CM contained IL-1α, IL-10, IL-11, M-CSF, OSM, SCF, VEGF, as well as a number of chemokines and other proteins. For a number of these proteins, we have already verified that the mRNA for them is expressed in the ES cells. This indicates that ES cells produce and secrete these cytokines. Some of these cytokines are known to have an enhanced survival/antiapoptosis effect on progenitors. IL-6, FGF-9, and TNF-a, which were not detected prior to irradiation of the ES cells, were seen after ES cells were irradiated. Irradiation of the ES cells enhanced release of some proteins and decreased release of others. ES cell CM also stimulated CFU-GM colony formation. Thus, undifferentiated murine ES cells growing in the presence of LIF produce/release a number of biologically active interleukins, CSFs, chemokines, and other growth modulatory proteins. Oct-4 is a marker for undifferentiated ES cells. We wondered if Oct-4 might be a key player for cytokines released from ES cells which supported CFU-GM survival and antiapoptosis. Oct-4 conditional knockout cell line ZHBtc4, received from Dr Austin Smith, was used. CM from the wild type ES cell line enhanced survival of CFU-GM similar to that of other ES cell lines, while the Oct-4 knockout ES cell line didn’t. These results indicate that release of proteins involved in survival enhancement may be related to Oct-4 expression. We also found that the wild type cell line which expressed Oct-4 didn’t initiate caspase 3 dependent apoptosis after mitotic stress. However ZHBTc4, the Oct-4 deleted cell line demonstrated caspase 3 dependent apoptosis. These results may be of physiological significance, although this has not yet been proven, and suggest the possibility of potential future applicability for use of irradiated ES cells as accessory cells for growth modulation in vitro and in vivo.

Description: Chemokines are a large family of cytokines that direct normal leukocyte migration. They also have been implicated in leukocyte development and in the pathogenesis of many diseases. The CC chemokine CCL21, also known as Exodus-2, SLC, 6Ckine, and TCA4 induces both the adhesion and migration of human T cells. CCL21 is hypothesized to regulate the trafficking of T cells through secondary lymphoid tissues. To test this hypothesis, a transgenic mouse model was generated that placed the expression of mouse CCL21 (mCCL21) under the control of the T cell-specific lck promoter to abrogate the concentration gradient to which T cells normally respond. Overexpression of mCCL21 in T cells resulted in defects in CCL21- and CCL19-induced T-cell chemotaxis, node T-cell subpopulations, and lymph node architecture. The regulation of T-cell trafficking in secondary lymphoid tissues by CCL21 is therefore a tightly regulated system that can be altered by changes in the level of environmental CCL21 protein.