Abstract 1068: Navigating next-generation pre-clinical models of drug discovery & development with RNA-seq

Propose: We evaluated the anti-leukemic effect of GSK3 inhibitor for specific type of MLL-tranlocated cell line. Materials and methods: l GSK3 inhibitor: LiCl, SB216763, GSK3-IX l Cell line: MV4-11 [human lymphoblast, t(4:11)] MOLM-13 [human acute myeloid leukemia, ins(9:11)(q23;p22p23)] l Cell proliferation assay: XTT assay, Clonogenic assay l GSK3i activity & detection of histone acetylation and H3K4 methylation: Immunoblotting Summary: MLL gene encodes an enzyme which methylates histone H3 DNA-binding protein and positively regulates the expression of target genes in normal tissue. MLL rearrangement which is found in acute myeloid leukemia (AML), especially therapy-related AML, encodes fusion proteins that have lost H3 lys4-specific (H3K4) methyltransferase activity, and causes a transformation of hematopoietic cells into leukemia stem cells. GSK3, a serine/threonine kinase, plays a key role in glycogen metabolism and is constitutively expressed in resting cell. Although it has been thought that the inhibition of GSK3 causes apoptosis in neurons and other tissues, a paradoxical and unexpected cell death effect of GSK3 inhibition were reported recently in leukemic cell line with MLL translocation, especially t(4;11). Regarding the mechanism of the cytotoxic effect of GSK3 inhibitor, various molecules associated with the signaling pathway around MLL gene have been investigated. Acetylation of H3 peptides stimulates the activity of SET domain in MLL. MLL gene is cleaved at 2 conserved sites by taspase-1; N-terminal 320 kD fragment (N320) and a C-terminal 180 kD fragment (C180). RNA interference-mediated knockdown of taspase-1 resulted in an unprocessed MLL, and finally a loss of proper HOX gene expression. However, the exact mechanism has been still unknown and it remains to be uncertain whether GSK3 inhibitor shows cell death effect on various leukemic cell lines of MLL translocation with other partner gene. We intended to determine whether the inhibition of GSK3 induced cell death in different types of leukemia cell lines with MLL translocation. We examined the apoptotic effects of GSK3 inhibitors on MV4-11 and MOLM-13 cell line. LiCl and GSK3-IX induced cell death, H3K4 methylation and histone acetylation in dose-dependent manners on MV4-11 but did not induce on MOLM-13. However, SB216763 did not induced cell death both cell lines. Conclusion: Cell death effects with the inhibition of GSK3 may be different among leukemic cell line with MLL translocation according to the partner gene of MLL translocation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5479.

TRAIL receptor agonists are attractive anti-tumor agents because of their capability to induce apoptosis in cancer cells by activating death receptors 4 and 5 (DR4 and DR5) with little toxicity against normal cells. We previously reported that GST-TRAIL efficiently induced cell death in breast cancer cells, particularly mesenchymal triple negative breast cancer (TNBC) – so called basal B breast cancer cells (Rahman et al., Adv. Cancer Res. 2009). Recently, a newly developed multivalent TRAIL receptor agonist designed to activate DR5, has been shown to be a TRAIL super-agonist with significantly enhanced potency in multiple cancer cell lines (Swers et al., Mol Cancer Ther. 2013). We hypothesized that MEDI3039, developed from this TRAIL super-agonist, is a potential new therapeutic agent to be used in human breast cancer treatment. As model systems, we used 19 breast cancer cell lines that can be categorized into 4 different groups: ER+, HER2 amplified, TNBC basal A and TNBC basal B. MEDI3039- or GST-TRAIL-induced cell death was analyzed by an MTS assay in 96 well format after 72h of treatment. MEDI3039- or GST-TRAIL-induced caspase activation was measured by Caspase-glo 3/7 assay. Z-VAD-FMK was used as a pan-caspase inhibitor. To verify the receptor for MEDI3039, siRNA against DR4 and DR5 were transfected to cells and tested in MTS assay and Western blotting. MEDI3039 induced cell death in MDA-MB231 (TNBC basal B), and the IC50 was 4.71pM. By contrast, GST-TRAIL induced cell death in this cell line with an IC50 of 624 pM (a 132 fold difference). MEDI3039 and GST-TRAIL induced cell death was completely inhibited by Z-VAD-FMK, indicating that cell death was the result of caspase-mediated apoptotic pathway. Knockdown of DR5, but not DR4, inhibited MEDI3039-induced cell death, demonstrating that MEDI3039-mediated apoptosis requires DR5. MEDI3039 induced cell death in multiple breast cancer cell lines, but the sensitivity varied between cell lines from the four different subtypes. TNBC basal B group was the most sensitive (avg IC50= 1.4 pM), TNBC basal A group was next most sensitive (avg IC50 = 203 pM, HER2 amplified group was less sensitive (avg IC50 = 314 pM), and ER+ group was the least sensitive to MEDI3039 (avg IC50= 403 pM). This was similar to what was observed with GST-TRAIL. Importantly, MEDI3039 was at least 2 orders of magnitude more potent compared with GST-TRAIL in most cell lines tested. Drug combination experiments indicated that MEDI3039 has synergistic effect with multiple drugs, including cisplatin, MK1775. Animal breast cancer xenograft experiments are planned to test the efficacy of MEDI3039 in vivo. Further work to identify biomarker(s) that correlate with MEDI3039 sensitivity, and effective combinations that enhance the toxicity of MEDI3039 especially in the resistant breast cancer subtypes are ongoing. In conclusion, MEDI3039 is a potent TRAIL receptor agonist in breast cancer cells and has potential as a cancer drug in breast cancer patients, especially those with TNBC basal B. Citation Format: Greer YE, Tice D, Lipkowitz S. MEDI3039, a novel highly potent tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) receptor agonist, induces apoptotic cell death in breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-06.

The involvement of the death adaptor protein FADD and the apoptosis-initiating caspase-8 in CD95 and TRAIL death signalling has recently been demonstrated by the analysis of the native death-inducing signalling complex (DISC) that forms upon ligand-induced receptor cross-linking. However, the role of caspase-10, the other death-effector-domain-containing caspase besides caspase-8, in death receptor signalling has been controversial. Here we show that caspase-10 is recruited not only to the native TRAIL DISC but also to the native CD95 DISC, and that FADD is necessary for its recruitment to and activation at these two protein complexes. With respect to the function of caspase-10, we show that it is not required for apoptosis induction. In addition, caspase-10 can not substitute for caspase-8, as the defect in apoptosis induction observed in caspase-8-deficient cells could not be rescued by overexpression of caspase-10. Finally, we demonstrate that caspase-10 is cleaved during CD95-induced apoptosis of activated T cells. These results show that caspase-10 activation occurs in primary cells, but that its function differs from that of caspase-8.

The Role of Iron in the Cytotoxicity Induced in Hematopoietic Malignant Cell Lines by an Anti-Transferrin Receptor IgG3-Avidin Fusion Protein and Gambogic Acid.

Fn14 is a therapeutic target in various diseases. Anti-Fn14 antibodies activate the alternative NFκB pathway but not other Fn14-related activities induced by soluble or membrane-bound TWEAK. FcγR-bound anti-Fn14 antibodies, however, activate the full spectrum of Fn14-associated activities. Anti-Fn14 antibodies elicit agonistic activities differing from those of the natural Fn14 ligand TWEAK. These findings influence the rationale of designing Fn14-targeted therapies. The Fn14-specific monoclonal antibodies PDL192 and P4A8, which are under consideration in clinical trials, showed no agonistic activity with respect to IL8 production and cell death induction. However, oligomerization with protein G or binding to Fcγ receptors converted both anti-Fn14 antibodies into potent agonists. TNF-like weak inducer of apoptosis (TWEAK), the ligand of Fn14, occurs naturally in two forms with partly different signaling capabilities, as a membrane-bound ligand and as a soluble trimeric molecule. Although membrane TWEAK strongly triggers all Fn14-associated pathways, soluble TWEAK predominately triggers the alternative nuclear factor κB (NFκB) pathway and enhances TNF-induced cell death but has only a poor effect on the classical NFκB pathway and chemokine production. Thus, the oligomerized and FcγR-bound anti-Fn14 mAbs mimicked the activity of membrane TWEAK. Notably, both anti-Fn14 antibodies significantly triggered p100 processing, the hallmark of the alternative NFκB pathway, and therefore resembled soluble TWEAK. In contrast to the latter, however, the anti-Fn14s showed no effect on TNF receptor 1-induced cell death and P4A8 even blocked the corresponding TWEAK response. Thus, we showed that Fn14 antibodies display an alternative NFκB pathway-specific agonistic activity but fail to phenocopy other activities of soluble TWEAK, whereas oligomerized or FcγR-bound Fn14 antibodies fully mimic the activity of membrane TWEAK. In view of the trivalent nature of the TWEAK-Fn14 interaction, this suggests that the alternative NFκB pathway is uniquely responsive already to Fn14 dimerization enabling antibodies to elicit an unnatural response pattern distinct from that of the naturally occurring Fn14 ligands.

Mild ischemic/hypoxic insult produces delayed neuronal damage in many brain areas which may undergo apoptosis. A phospholipid, PAF, is thought to mediate neuronal damage produced by ischemia/reperfusion, PAF production and metabolism are increased by ischemic/hypoxic lesion, and PAF antagonists are shown to protect neuronal damage. However, the molecular mechanism of PAF action on the neurons subjected to hypoxic/ischemic insult is unclear because of the heterogeneity of brain. Therefore, the possible role of PAF receptor(s) in the ischemia/reperfusion was examined in order to further our knowledge concerning the underlying mechanisms of neuronal damage due to ischemic/hypoxic insult. Specifically, experiments were designed to investigate the effects of hypoxia/hypoglycemia in a hippocampus-derived cell line, HN33.11 cells, a cell line which exhibits PAF-stimulated PI metabolism via Gq and Gi proteins on (1) the role of PAF receptors in cell death due to hypoxia/ hypoglycemia and (2) functions of PAF receptor-coupled G proteins in mediating cellular damage during hypoxic insult. A delayed cell death with apoptotic features was observed when cells were treated with hypoxia/hypoglycemia condition (ischemia) followed by incubation in normal control medium (reperfusion). PAF produced in the early reperfusion elicited cellular death which was inhibited by PAF receptor antagonists. The PAF receptor-elicited intracellular Ca²⁺ increase may mediate the PAF action during reperfusion. Treatment with pertussis toxin increased cellular damage induced by ischemia/reperfusion. PTX treatment also potentiated cell death induced by the PAF analogue. On the other hand, G[alpha]q antisense treatment protected cells from ischemia/reperfusion induced cell death by decreasing G[alpha]q protein levels. Moreover, this treatment also attenuated MC-PAF-induced cell death during ischemia/reperfusion. In conclusion, hypoxia/hypoglycemia in HN33.11 cells induces a delayed cell death via apoptosis; this is mediated by a delayed increase in PAF production and activation of PAF receptor. Gq and Gi proteins, which are functionally coupled to PAF receptor(s), play antagonistic roles in PAF receptor mediated ischemia/ reperfusion-induced cellular damage. The predominant coupling between PAF receptor and Gq may underlie the injury induced by ischemia/reperfusion in HN33.11 cells.

To evade the immune system, the etiologic agent of plague, Yersinia pestis, injects an exceptionally active tyrosine phosphatase called YopH into host cells using a type III secretion system. We recently reported that YopH acutely inhibits T cell antigen receptor signaling by dephosphorylating the Lck tyrosine kinase. Here, we show that prolonged presence of YopH in primary T cells or Jurkat T leukemia cells causes apoptosis, detected by annexin V binding, mitochondrial breakdown, caspase activation, and internucleosomal fragmentation. YopH also causes cell death when expressed in HeLa cells, and this cell death was inhibited by YopH-specific small molecule inhibitors. Cell death induced by YopH was also prevented by caspase inhibition or co-expression of Bcl-xL. We conclude that YopH not only paralyzes T cells acutely, but also ensures that the cells will not recover to induce a protective immune response but instead undergo mitochondrially regulated programmed cell death.

JX06, a Novel PDK1 Inhibitor, Induces Myeloma Cell Apoptosis By Metabolic Reprogramming and Works Synergistically with Bortezomib

Multiple myeloma (MM) is a neoplastic proliferation of plasma cells and remains an incurable disease because of the development of drug resistance. Histone deacytylase (HDAC) inhibitors are a new class of chemotherapeutic reagents that cause growth arrest and apoptosis of neoplastic cells. Depsipeptide, a new member of the HDAC inhibitors, was found to be safe in humans and has been shown to induce apoptosis in various cancers. In order to evaluate the effects of depsipeptide, a MM cell line, U266 [interleukin (IL)-6 dependent], was analysed for viability and apoptosis. The combined effect of depsipeptide with melphalan and changes in BCL-2 family proteins (BCL-2, BCL-XL, BAX and MCL-1) were also investigated. In addition, the RPMI 8226 cell line (IL-6 independent), and primary patient myeloma cells were also analysed for apoptosis after depsipeptide treatment. Depsipeptide induced apoptosis in both U266 and RPMI 8226 cell lines in a time- and dose-dependent fashion, and in primary patient myeloma cells. We also demonstrated that depsipeptide had an additive effect with melphalan (10 micromol/l). BCL-2, BCL-XL and MCL-1 showed decreased expression in depsipeptide-treated samples. Based on recent clinical trials demonstrating minimal clinical toxicity, our study supports the future clinical utilization of depsipeptide in the management of MM.

Bak is a prototypic pro-apoptotic Bcl-2 family protein expressed in a wide variety of tissues and cells. Recent studies have revealed that Bcl-2 family proteins regulate apoptosis as well as autophagy. To investigate whether and how Bak exerts a regulatory role on autophagy-related events, we treated independent cell lines, including MN9D neuronal cells, with nigericin, a K(+)/H(+) ionophore. Treatment of MN9D cells with nigericin led to an increase of LC3-II and p62 levels with concomitant activation of caspase. Ultrastructural examination revealed accumulation of autophagic vacuoles and swollen vacuoles in nigericin-treated cells. We further found that the LC3-II accumulated as a consequence of impaired autophagic flux and the disrupted degradation of LC3-II in nigericin-treated cells. In this cell death paradigm, both transient and stable overexpression of various forms of Bak exerted a protective role, whereas it did not inhibit the extent of nigericin-mediated activation of caspase-3. Subsequent biochemical and electron microscopic studies revealed that overexpressed Bak maintained autophagic flux and reduced the area occupied by swollen vacuoles in nigericin-treated cells. Similar results were obtained in nigericin-treated non-neuronal cells and another proton ionophore-induced cell death paradigm. Taken together, our study indicates that a protective role for Bak during ionophore-induced cell death may be closely associated with its regulatory effect on maintenance of autophagic flux and vacuole homeostasis.

Fangchinoline is a novel anti-tumour agent with little known of its cellular and molecular mechanisms of action. Here we have investigated the mode of cell death induced by fangchinoline and its underlying mechanism in two human hepatocellular carcinoma cell lines, HepG2 and PLC/PRF/5. Apoptosis and autophagy were monitored in fangchinoline-treated HepG2 and PLC/PRF/5 cells by histological methods. The signal transduction pathways involved in activation of autophagy were examined, using immunoblotting, real-time PCR and siRNA techniques. Fangchinoline did not induce apoptosis in HepG2 and PLC/PRF/5 cells but triggered, dose-dependently, autophagy, an alternative mode of cell death which may contribute to fangchinoline's anti-tumour action. Nuclear translocation of p53 was involved in induction of autophagy by fangchinoline, followed by selective transactivation of the autophagy-related gene sestrin2 and initiation of the autophagic process. Signalling by the AMP-activated protein kinase was also involved as a downstream target of sestrin2 and induced mTOR-independent autophagic cell death in both cell lines. siRNA for Atg 5 or pharmacological block of p53 abolished fangchinoline-induced autophagy and inhibition of autophagy switched cell death to apoptosis in these cells, suggesting that cell death is irreversible once autophagy is induced by fangchinoline. Fangchinoline is a highly specific agent inducing autophagic cell death in hepatocellular carcinoma cells with a novel mechanism, which elucidates the potential of fangchinoline to potentiate programmed cell death in cancer cells.

Background: Checkpoint kinase 1 (Chk1) is a serine/threonine protein kinase that regulates cell division in response to genotoxic stress by arresting cell cycle progression in the S & G2 phases. Pharmacological inhibition of Chk1 is proposed to target tumor cells with increased DNA replication stress, resulting in the uncoupling of DNA replication checkpoint function and the induction of DNA damage and cell death. These properties make Chk1 inhibition a novel therapeutic approach as a single agent in cancers with high replication stress that is driven by oncogenic signaling and loss of parallel DNA damage response pathway function. Methods and Results: This report highlights the activity of the orally bioavailable, selective small molecule Chk1 inhibitor, CASC-578, in solid tumor derived cell lines. CASC-578 is a sub-nanomolar enzymatic inhibitor of Chk1 with limited off-target activity against a panel of protein kinases. When evaluated in a large cell line panel in vitro, CASC-578 demonstrated a broad potency range as a single agent in solid tumor derived cells lines, with IC50s ranging from 30 nM to greater than 50 μM. Several solid tumor types demonstrated enriched sensitivity to CASC-578 in vitro, including gastric, non-small cell lung and ovarian cancers. Treatment of sensitive cell lines with CASC-578 resulted in the induction of DNA damage, as measured by phosphorylated histone H2AX, and the induction of cell death. CASC-578 was active as a single agent in SK-MES-1 and NCI-H727 NSCLC tumor xenograft models in vivo with minimal effects on body weight in treated mice. In addition to the potent single agent activity of CASC-578, combination with the Wee1 inhibitor AZD-1775 was highly synergistic in vitro in multiple solid tumor cell lines and the combination was more efficacious than either agent alone in NSCLC tumor xenograft models. These data support the advancement of CASC-578 into clinical development as a potential therapeutic agent for the treatment of solid tumor diseases. Experiments are ongoing to identify biomarkers associated with sensitivity to CASC-578 as a single agent in solid tumor cell lines to prospectively identify tumor genotypes that are more responsive to the drug. Citation Format: Alex Vo, Janelle Taylor, Robert Rosler, Julia Piasecki, Dina Leviten, Teresa Sierra, Ashley Dozier, Kevin Klucher, Bob Boyle, Rich Boyce, Scott Peterson. CASC-578, a novel Chk1 inhibitor, is active as a single agent in solid tumors and displays synergistic anti-tumor activity in combination with Wee1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 295. doi:10.1158/1538-7445.AM2017-295

PIM kinases (PIM1,2, and 3) are constitutively active serine/threonine protein kinases which regulate cell cycle, survival, and drug resistance and have been strongly implicated in cancer cell survival and tumorigenesis. Although expression levels of PIM family members (PIM1, 2, and 3) differ by cancer type, PIM1 and PIM2 kinases are preferentially overexpressed in hematological malignancies. Here we studied the biological effects of treatment with a novel pan-PIM inhibitor, JP_11646 exhibiting potent inhibition of PIM2 kinase activity, in human acute myeloid (AML), acute lymphocytic leukemia (ALL), and lymphoma cell lines. Human AML cell lines (THP-1, KG-1, HL60-VCR, ML2, MV(4;11)) were evaluated for basal mRNA and protein expression of PIM kinases using real time PCR and western blot analysis, respectively. A panel of thirty additional human AML, ALL (Nalm-6), and lymphoma (RAJI) cell lines were exposed to JP_11646 at concentrations ranging from 0.001 to 10 uM for 48-72 hours followed by measurements of cell proliferation by MTT assay and cell viability by CellTiter-Glo assay. The Hill model was used to fit each concentration-response curve for each cell line and endpoint. Nonlinear regression analysis was performed using Phoenix 64 for estimation of IC50 and Imax. AML (HEL) cells after 72 hour of JP_11646 treatment were analyzed for induction of apoptosis measured via annexin V and 7AAD flow cytometry. AML cell lines exhibited differential basal mRNA expression of PIM kinases. The cell lines that expressed the highest levels of PIM1, PIM2, and PIM3 mRNA were THP-1 and ML2, respectively. Basal expression of the PIM2 protein revealed the presence of all three PIM2 isoforms in AML (HEL, THP-1) cells. JP_11646 treatment resulted in inhibition of proliferation across the entire panel of AML and ALL cell lines, with the overall potency (IC50) ranging from 0.07 to 0.37 uM and maximal inhibition ranging from 70 to 97.5 percent. As the drug exposure time increased from 48 to 72 hours, the maximal inhibition increased 2 to 3-fold. Inhibition of cell viability across a panel of 30 leukemia and lymphoma cell lines demonstrated a similar IC50 range from 0.05 to 0.25 uM. AML (HEL) cells treated with JP_116464 at doses of 0.1 and 0.5 uM for 72 hours exhibited a concentration-dependent induction of apoptosis of 32% and 89% percent, respectively. Taken together, our results demonstrate broad anti-leukemic activity and potency of the novel PIM kinase inhibitor, JP_11646, across a wide panel of human AML and ALL cell lines with induction of apoptosis and cell death at nM concentrations. Further, investigation of the in vivo effects of JP_11646 in representative human AML xenograft models is ongoing. Citation Format: Krista E. Pundt, Carmen M. Baldino, Justin Caserta, Stephane Dumas, Yvonne Flanders, Eunice S. Wang, Gerald J. Fetterly. Preclinical efficacy of the novel PIM2 kinase inhibitor, JP_11646, in human acute leukemia models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5459. doi:10.1158/1538-7445.AM2014-5459

The failure of drug efficacy in clinical trials remains a big issue in cancer research. This is largely due to the limitations of two-dimensional (2D) cell cultures, the most used tool in drug screening. Nowadays, three-dimensional (3D) cultures, including spheroids, are acknowledged to be a better model of the in vivo environment. Immunogenic cell death (ICD) has the potential to be exploited in immunotherapy by activating the host’s own immune system against the cancerous cells, but analysis of ICD induction (including ferroptosis) in 3D cultures has not been performed so far. In this work, we have optimized cell death induction in tumor spheroids. Our cell death analysis method (3DELTA) uses Sytox dyes as a cell death marker and Triton X-100, which efficiently permeabilizes all cells in spheroids, was used to establish 100% cell death. The 3DELTA method was able to detect cell death type without the need to disaggregate spheroids. Moreover, in this work we also demonstrated that 2D experiments cannot be extrapolated to 3D cultures as 3D cultures are less sensitive to cell death induction. Ferroptotic cell death induction in spheroids lead to 50% of cell death whereas in 2D cultures, this caused 90% of cell death. The tumor microenvironment is an important factor that can affect the outcome of anti-cancer therapy. By adding endothelial cells (EC) to cancer cells (CC) in an optimized ratio (EC:CC), we were able to form vascularized spheroids. In conclusion, these spheroids will be a more relevant model for in vivo tumors by analyzing interactions between the different cell types and can be further used for analysis of their immunogenic potential.

VDAC's role in Bak‐dependent mitochondrial outer membrane permeabilization (MOMP) is controversial. Gordon Shore makes sense of it all in light of an EMBO reports paper that shows that VDAC mediates mitochondrial recruitment of Bak, thereby controlling tBid‐induced MOMP.