ALBERT

Description: Background The epitome of cancer treatment personalization is N=1 segmentation where a custom therapy is designed for every patient. Because most cancer aberrations are not actionable mutations and tumors can have more than one actionable mutation, this one biomarker/one drug approach to cancer personalization has inherent limitations due to its over simplification. Personalization 2.0 methodology creates a patient simulation avatar incorporating a patient’s genomic profile information holistically. Methods Bone marrow samples from two myeloma patients (P1 and P2) refractory to most recent treatment was collected, and P1’s sample was sorted into CD138+ and CD138- cells. The patient cells were analyzed for chromosomal alterations using Comparative Genomic Hybridization (aCGH) arrays by GenPath Diagnostics and cytogenetic chromosome analysis by Washington University School of Medicine and New York University (NYU), respectively. Using this information, a predictive simulation avatar model of each patient was created by Cellworks based on genomic profile of patients. A digital functional library of over 80 FDA-approved drugs and agents currently in clinical trials were simulated individually and in combination using the two patient avatars to create a personalized treatment for each patient. The findings were prospectively validated using patient cells ex vivo as assessed by MTT assay at New York University. Results P1 aberrations included trisomy of CCND1 and deletion of TP53 along with single copy losses in different arms of chromosomes 1, 6, 8, 12, 13, 14, 16, 17 and 22 and gains in different arms and regions of chromosomes X, 1, 4, 7, 9, 17, 3, 5, 11, 15 and 19, indicating the presence of hyperdiploid clones. Using this information, 897 gene perturbations were included to model this patient simulation avatar. Simulation predicted high beta-catenin (CTNNB1) activity with increased hedgehog and NOTCH pathways that were inherent causes of Bortezomib resistance. Significant activation of STAT3 and STAT5 due to amplification of IL6 pathway, JAK2 and JAK3 was noted. Amplifications of MET, IGFR and FGFR converged at ERK and AKT signaling loops. Along with deletion of TP53, this profile had amplification of many anti-apoptotic genes including survivin, MCL1 and XIAP. Modeling predicted sensitivity to the JAK inhibitor Tofacitinib, a drug approved for rheumatoid arthritis. This was prospectively validated ex vivo, and the experimental data correlated with the prediction showing a reduction in viability. P2 aberrations include losses in chromosomes X and 9 and a chromosome 11:14 translocation that is a common occurrence in MM. This translocation results in an amplification of CCND1 expression. The genomic aberrations reported include knockdown of tumor suppressors RXRA, TGFBR1, TJP2 and TSC1. TSC1 regulates the mTOR pathway, and its deletion causes an aberrant activation of mTOR and its downstream targets. Reduced expression of RXRA and TJP2 both in different manners leads to increase in AP1 activation. NFkB is also activated due to RXRA reduction. TGFBR1 reduction decreases the expression of cell cycle inhibitors via SMAD2/3 down-regulation. In this patient avatar, modeling predicted sensitivity to a combination of Sirolimus and Trametinib. Ex vivo validation confirmed this prediction of additive synergy of these two drug agents in the context of this patient. Conclusions This study demonstrates and validates the personalization of treatment through two patient cases based on creating predictive simulation avatar models using genomic profile information. This modeling holistically incorporates all genomic aberration information and is not limited to associating drugs to actionable mutations. Disclosures Doudican: Cellworks: Research Funding. Vali:Cellworks: Employment. Basu:Cellworks: Employment. Kumar:cellworks: Employment. Singh:Cellworks: Employment. Sultana:Cellworks: Employment. Abbasi:Cellworks: Employment, Equity Ownership.

Description: Introduction Development of resistance to single agent therapy is a significant clinical obstacle in the treatment of multiple myeloma (MM). Genetic mutations and the bone marrow micro-environment are major determinants of MM resistance mechanisms. Given the complexity of MM, the need for combinatorial therapeutic regimens targeting multiple biological mechanisms of action is pressing. Repurposing has the advantage of using drugs with known clinical history. Methodology We used a predictive simulation-based approach that models MM disease physiology in plasma cells by integrating and aggregating signaling and metabolic networks across all disease phenotypes. We tested the efficacy of over 50 repurposed molecularly targeted agents both individually and in combination across simulation avatars of the MM cell lines OPM2 and U266. OPM2 harbors mutations in KRAS, CDKN2A/2C, PTEN, RASSF1A and P53, whereas U266’s mutational components include BRAF, CDKN2A, P53, P73, RASSF1A and RB1. These cell lines were used as models because they possess mutations in genes classically known to be involved in myeloma. The predicted activity of novel combinations of existing drug agents was validated in vitro using standard molecular assays. MTT and flow cytometry were used to assess cellular proliferation. Western blotting was used to monitor the combinatorial effects on apoptotic and cellular signaling pathways. Synergy was analyzed using isobologram plots and the Bliss independence model. Results Through simulation modeling, we identified two novel therapeutic regimens for MM using repurposed drugs: (1) AT101 (Bcl2 antagonist) and tesaglitazar (PPAR α/γ agonist) and (2) Ursolic acid (UA, inhibitor of NFκβ) and SP600125 (pan-JNK inhibitor). Simulation predictions showed that combining the IC30 concentrations with respect to viability of AT101 and tesaglitazar reduced proliferation by 40% and viability by 50%. Similarly simulation predictions showed that the combination of the IC30 concentrations of UA and SP600125 reduced proliferation by 50% and viability by 40%. Corroborating our predictive simulation assays, 10 µM tesaglitazar and 2 µM AT101 caused minimal growth inhibition as single agents in OPM2 and U266 MM cell lines. Growth inhibition in these cell lines is synergistically enhanced when the drugs are used in combination, reducing cellular viability by 88% and 77% in OPM2 and U266 cells, respectively. Similarly, proliferation was reduced by 34% with 7.5 μM UA and 25% with 10 μM SP600125 in OPM2 cells. When used in combination, cellular proliferation was synergistically reduced by 64%. In addition, isobologram analysis predicted synergy of lowered doses of the drugs in combination. Both combinations synergistically inhibited proliferation and induced apoptosis as evidenced by an increase in the percentage sub-G1 phase cells and cleavage of caspase 3 and poly ADP ribose polymerase (PARP). Conclusions These results highlight and validate the use of our predictive simulation approach to design therapeutic regimens with novel biological mechanisms using drugs with known chemistries. This allows for design of personalized treatments for patients using their tumor genomic signature beyond the “one-gene, one-drug” paradigm. The reuse of existing drugs with clinical data facilitates a rapid translational path into clinic and avoids the uncertainties associated with new chemistry. The corroboration of these results with patient derived cell lines will be pursued and discussed. Disclosures: No relevant conflicts of interest to declare.

Description: Background The unique signature of a patient’s tumor mandates the need to rationally design personalized therapies employing N=1 segmentation conceptually. Repurposing of existing drug agents with validated clinical safety and pharmacokinetics data provides a rapid translational path to clinic which otherwise would require years of development time and associated new chemical risks. By focusing on rationally designed personalized treatment mechanisms, our strategy targets multiple key pathways to address the clinical problem of emergence of single therapy resistance. In order to overcome MM resistance, we have (1) employed predictive simulation modeling based upon patient genetic and environment profiling to design patient context specific combinatorial therapeutic regimens using library of drug agents from across indications with prior clinical data and (2) validating designed therapy ex-vivo in patient derived cell lines. Methods Clinical patient samples were analyzed for chromosome evaluation and molecular cytogenetic analysis by NYU. Using this information, an in silico simulation avatar of the patient was created. To identify effective personalized therapeutics, we focused our study on compounds from the National Center for Advanced Translational Science (NCATS) and other molecularly targeted agents. The predictive simulation based approach from Cellworks provides a comprehensive representation of MM disease physiology incorporating signaling and metabolic networks with an integrated phenotype view. This extensively validated simulation model predicts clinical outcomes with phenotype and bio-marker assays. Hits were shortlisted from over thousand pharmacodynamic dose-response simulation studies using criteria of efficacy and synergy. Computer modeling predicted that therapeutic combination mechanistically targets apoptotic pathways and the combination of the agents provides greater than additive activity. These predictive findings are in the process of being assessed ex vivo and retrospectively validated. Results The analysis detected loss of chromosome 13 signal consistent with monosomy 13 and loss of TP53 signal consistent with deletion of TP53; all other probes contained normal signal patterns. The shortlisted therapeutic combination identified from predictive simulation-based screening was BEZ235 (PI3K/mTOR inhibitor) and ABT-199 (BCL2 inhibitor). IC30 concentrations of the single agents resulted in a 56% inhibition of proliferation and 49% inhibition of viability in predictive simulations. The apoptotic markers CASP3, CASP9, Cleaved-PARP1 and BAK1 increased by 74% (1.75 fold), 132% (2.32 fold), 81% (1.8 fold), 217% (3.17 fold), respectively. The proposed mechanism of action using simulation model identified the p53 deletion as responsible for increased BCL2 activity and levels of activated AKT. Deletion of p53 increased levels of activated AKT via decreases in PTEN and IGFBP3. Hence, a mechanism that targets the PI3K/AKT/mTOR and BCL2 family showed efficacy in the simulation avatar of the patient and are currently being validated ex-vivo in patient cells. Conclusions This study demonstrates and validates simulation approaches and technologies to leverage big data from patient genomic analysis to create a simulation avatar for rational design of personalized therapeutics. This level of personalization, beyond linking point mutations to associated drugs targeting the same mutations, truly incorporates the broad patient tumor signature in translational path forward. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 5012 Development and progression of multiple myeloma is dependent on the bone marrow (BM) microenvironment. Bone marrow stromal cells (BMSC) secrete Wnt ligands that activate the Wnt signaling pathway. The canonical Wnt pathway, which is mediated through the key transcriptional effector β-catenin (β-cat), is commonly deregulated in many cancers. Cells with β-cat-regulated transcription (CRT) are protected against apoptosis; conversely inhibition of CRT may inhibit cell proliferation. In this study we tested the efficacy of recently described inhibitors of CRT (iCRTs) for their selective antagonistic effect on Wnt-β-cat in MM cells. Although, earlier studies have documented Wnt signaling in human MM cells, in order to test the chemosensitivity of iCRTs in myeloma cells, we first confirmed the expression of β-cat in human MM cell types. An immunofluorescence detection of β-cat in U266 cells showed nuclear localization in 〉 70% of the cells, a similar trend of nuclear β-cat was observed in MM1 and patient derived BMMC cells. This observation is consistent with the Western blot analysis of the total protein from three cell types. The above data on the expression of nuclear β-cat in MM cell lines and cells from patient sample (n=16) provides the rationale for using these cells to test the efficacy of iCRTs (Oxazole-iCRT-3 and Thiazole-iCRT-5), that are designed to target β-cat signaling. Wnt reporter plasmid STF16-transfected MM cells treated with iCRTs at the doses of 10, 25, and 50 μM showed a dose dependent decrease (2–3 fold) in the Wnt/β-cat reporter activity, with a significant decline at a maximum dose of 50 μM (p3 fold determined by qRT-PCR analysis. To determine whether the effect of iCRTs on the VEGF activity is specific to its ability to antagonize b-cat activity, we used culture medium collected from cells transiently transfected with siRNA for β-cat. siRNA mediated down regulation of β-cat showed a decrease in the VEGF levels, thereby confirming that the effect of iCRTs is indeed mediated by their inhibitory effect on b-cat. Although several aspect of our key findings are yet to be confirmed in preclinical in vivo models for MM, this part of the study provide evidence that indicate a VEGF-dependent increase in cell proliferation and migration of MM cells that can be antagonized by specific inhibitors of nuclear b-cat, thereby underscoring the importance of developing iCRTs as a novel class of Wnt-directed therapeutics in human MM. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 5140 Persistent paraprotein production in plasma cells necessitates a highly developed rough endoplasmic recticulum (ER) that is exquisitely sensitive to perturbations in protein synthesis. Targeting ER stress- related signaling has been clinically validated in the treatment of multiple myeloma (MM) as evidenced by the response to treatment with bortezomib (BTZ). Despite impressive response rates, BTZ carries the potential for serious side effects, and the development of resistance to BTZ is a clinical issue. We therefore sought to identify novel drug combinations that effectively generate ER stress. Here, we report that sulforaphane, a naturally occurring isothiocyanate found in cruciferous vegetables, synergistically enhances the cytotoxicity of arsenic trioxide (ATO), an agent that has shown clinical activity in MM, in a panel of MM cell lines. As single agents, both 1 μM sulforaphane and 0.5 μM ATO have a modest effect on cellular proliferation in a panel of MM lines. However, when the agents are administered in combination, cellular proliferation is dramatically reduced. For example, in PCNY-1 MM cells, 1 μM sulforaphane has no effect and 0.5μM ATO causes a 29% reduction in proliferation. However, when administered together, the agents enhance growth inhibition to 73%, with a CI of 0.632 indicative of synergy. Four out of 5 MM cell lines tested displayed sulforaphane and ATO synergy. Combination treatment resulted in enhanced apoptotic induction as demonstrated by cleavage of PARP. Enhanced induction of ER stress signaling and activation of the unfolded protein response (UPR) upon combination treatment was demonstrated by enhanced expression of the molecular chaperone HSP90 along with increased phosphorylation of PERK (an ER transmembrane kinase and proximal effector of the UPR) and eIF2 (translational initiation factor). Additionally, increased splicing of XBP1 (a transcription factor of UPR target genes) was apparent upon combination treatment as compared to treatment with either agent alone. Our results show that sulforaphane can synergistically sensitize MM cells to the cytotoxic effects of ATO through promotion of ER stress generating mechanisms. Based upon these promising results, further evaluation of this safe, natural product as an ATO sensitizer in a clinical trial of MM patients is warranted. Additionally, this approach holds the promise as a means to identify and clinically validate natural products effective in the treatment of MM and/or inhibition of progression of asymptomatic MM. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 5021 Background: A distinguishing characteristic of myeloma plasma cells is the large quantity of paraprotein that they synthesize and secrete, rendering them especially sensitive to the effects of endoplasmic reticulum (ER) stress. Consistent with this notion, the proteasome inhibitor bortezomib disrupts protein equilibrium in the ER by preventing misfolded proteins from being properly degraded. Given the clinically validated importance of targeting ER stress mediated pathways in the treatment of multiple myeloma (MM), we sought to identify natural products that modulate pathways known to be an effective therapeutic target for MM for potential use to inhibit progression of asymptomatic MM to symptomatic MM without the limiting side effects of current targeted therapies. Methods: Using decreased protein processing in the secretory pathway as a measurable hallmark of ER stress, our screen employed the naturally secreted Gaussia luciferase (Gluc) as a reporter that can be easily monitored through extracellular release of luciferase activity in real time. KMS11 and ARP-1 MM cells expressing Gluc were exposed to compounds in our natural products library in order to identify those which potentially induce ER stress as measured by inhibition of Gluc secretion. The growth inhibitory activity of theaflavin-3, 3'–digallate (TF3) was further characterized by MTS assay. Mechanistic studies of ER stress related pathways including the unfolded protein response (UPR) and apoptotic cascades were analyzed by standard Western blotting techniques. Results: Our screen identified the black tea polyphenol TF3 as a significant inhibitor of GLUC secretion in ARP-1 and KMS-11 cells. TF3 at 0. 5 μM inhibits GLUC secretion by 73 and 68% in ARP-1 and KMS-11 cells, respectively. This inhibition observed is on par with that observed for bortezomib and tunacamycin (a well known inducer of ER stress). TF-3 effectively inhibits cellular proliferation and induces apoptosis in a panel of MM cell lines at physiologically achievable concentrations. Apoptotic induction is at least partially mediated by ER stress mediated pathways as upregulation of the protein chaperone HSP90 and phosphorylation of eIF2-a, a key mediator of the UPR pathway, occurs prior to caspase and PARP cleavage. Conclusions: Our results suggest that TF-3 inhibits protein secretion and MM cell growth through promotion of ER stress generating mechanisms. Based upon these promising results, further mechanistic evaluation and characterization of this safe, natural product as a prophylactic agent in the treatment of asymptomatic conditions like monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) is warranted. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 5022 Background: The extremely long development time and low success rate for new drug development programs has translated into limited clinical options in the treatment of cancer. The challenge is further compounded by drug resistance seen in clinical settings for approved standard of care therapeutic options like chemotherapy and targeted drugs like Bortezomib in Multiple Myeloma (MM). This has accelerated the need for initiatives and strategies which promote innovation but drastically reduce drug development failures through prediction of clinical outcomes. We present here a methodology and rationally designed therapeutic program for MM. This program has novel first-in-class mechanism of action and has been developed using a strategy of re-purposing and combinations based on National Center for Advanced Translational Sciences (NCATS) library of industry-provided drugs. Methods: The therapy design and development was completed using validated proprietary technology from Cellworks which enables simulation of cancer disease physiology computationally for predicting clinical outcomes. The comprehensive integrated representation of signaling and metabolic networks across all disease phenotypes and functional proteomics abstraction facilitated a large number of predictive studies in weeks with quantitative transparency into the network. The predictive cancer technology was customized to four MM profiles: OPM2, RPM1, SKMM2 and U266. Ten of the molecularly targeted drugs from the NCATS library were digitally screened alone and in combinations of two across the four MM profiles at four concentrations (C, C/2, C/4, and C/8). Based on screening of over six thousand predictive studies, three designed therapeutics hits were intelligently shortlisted based on synergistic impact on viability and proliferation. Results: The focused therapeutic candidate hit was selected based on synergy using viability endpoints and apoptosis markers such as caspase 3, PARP1 at sub-therapeutic doses. The proposed therapeutic regimen is a sub-therapeutic combination of AT101 and AVE0847. AT101 is a BCL2 inhibitor and AVE0847 is a PPAR alpha and gamma agonist agent. The concentration of each drug used in the designed therapy was close to IC30. The predictive results showed a synergistic increase in apoptotic markers caspase 3 and PARP1 cleaved form. This hit candidate is being pre-clinically validated experimentally across four MM profiles and results will be presented. Conclusion: The design of first in class mechanism of action based therapeutic strategies of drug rescue/repurposing has been used to identify AT101 and AVE0847 as a promising drug combination for use in MM which predicatively results in a synergistic increase in apoptosis. These results are currently being validated in vitro. The inherent multi target mechanism of action predicted by this combination could potentially eliminate the drug resistance challenges of single target therapeutics. Disclosures: No relevant conflicts of interest to declare.