Supplementary Table 1. Cell line and growth media information.

Abstract Background: PEEL-224 is a multivalent polymeric prodrug of the topoisomerase I inhibitor SN22 designed to sustain intratumoral exposure and reduce transporter-mediated efflux. Desmoplastic small round cell tumor (DSRCT) and osteosarcoma (OS) are high-risk sarcomas of children and adolescents/young adults where irinotecan+temozolomide (I/T) provides modest, short-lived benefit. Early-phase clinical evaluation of PEEL-224 is ongoing (NCT06709495, NCT06721689, NCT05329103). However, the comparative activity of PEEL-224±TMZ versus irinotecan-based therapy has not been defined in disease-relevant models, prompting evaluation in DSRCT and OS patient-derived xenografts (PDXs). Methods: PDX-bearing NSG mice were randomized to vehicle, irinotecan, I/T, PEEL-224, or PEEL-224+TMZ. Tumor growth was assessed using Vardi’s test for area-under-the-curve comparisons. Event-free survival (EFS) was defined as time to progression (≥100% relative tumor volume [RTV] increase from baseline) or euthanasia for tumor burden and analyzed by Kaplan-Meier with log-rank tests. Response criteria: PD = ≥100% RTV increase or euthanasia; SD = <100% increase and ≤50% reduction; PR = >50% reduction; CR = >95% reduction. Objective response rate (ORR) was the proportion achieving PR or CR. Treatment arms were expanded in an adaptive manner based on disease control to increase cohort size for key comparisons. Results: In DSRCT, tumor volume comparisons showed significantly greater control with PEEL-224 monotherapy vs irinotecan (p=0.04), while PEEL-224+TMZ and I/T showed similar control at this stage; cohort expansion is ongoing. EFS analysis showed PEEL-224 significantly prolonged EFS vs irinotecan (p=0.01) and PEEL-224+TMZ vs I/T (p=0.02). At end of therapy (Day 29), ORR was 0% (vehicle), 20% (irinotecan, 1/5 PR), 20% (I/T, 1/5 PR), 100% (PEEL-224, 5/5 PR), and 100% (PEEL-224+TMZ, 5/5 PR). At end of study (Day 113), irinotecan and I/T had 0% ORR, while PEEL-224 maintained 60% (3/5 PR, 2/5 SD) and PEEL-224+TMZ 100% (5/5 PR). Regimens were well tolerated. In OS, PEEL-224±TMZ produced disease stabilization and early regression, though statistical significance has not yet emerged (n=3/arm); expansion is ongoing. Conclusions: PEEL-224 demonstrated superior disease control as monotherapy and in combination over irinotecan-based therapy in DSRCT and early activity in OS. These preclinical data, alongside ongoing clinical evaluation, support advancement of PEEL-224 for high-risk pediatric and AYA sarcomas. Citation Format: Filemon S. Dela Cruz, Kristina C. Guillan, Samantha Brosius, Armaan H. Siddiquee, Glorife Ibanez Sanchez, Daoqi You, Kristen Victor, Paul Calder, Trent Fowler, Joshua D. Schiffman, Andrew L. Kung. Preclinical evaluation of PEG-[SN22]4 (PEEL-224), a multivalent polymeric camptothecin prodrug, in pediatric solid tumor patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1157.

Trastuzumab deruxtecan (T-DXd) is an ERBB2/HER2-targeting antibody-drug conjugate (ADC) with efficacy across adult cancers exhibiting variable HER2 expression. Prior studies demonstrating HER2 expression in osteosarcoma motivated a clinical trial of T-DXd in pediatric and adolescent/young adults with osteosarcoma, but the trial was terminated early because of inactivity. We evaluated the activity of T-DXd using osteosarcoma patient-derived xenograft (PDX) models and found a 22% objective response rate despite no detectable HER2 expression across PDXs tested. To further assess non-HER2-mediated activity, we evaluated the activity of T-DXd across 31 pediatric cancer cell lines and found osteosarcoma to be amongst the most resistant to T-DXd, as well as unconjugated deruxtecan, providing a potential explanation for the negative results observed in the clinical trial of T-DXd in osteosarcoma. T-DXd evaluation in PDX models representing pediatric histologies with greater intrinsic sensitivity to deruxtecan, including pediatric renal tumors and desmoplastic small round cell tumor, revealed both HER2-enhanced activity as well as substantial non-HER2-mediated activity, as evidenced by equipotent activity using an isotype-matched control ADC. Together, these results underscore translational opportunities for ADC therapeutics in tumor histologies with high sensitivity to the payload and in which enhanced tumor delivery may be mediated by antibody-targeted mechanisms as well as macromolecular characteristics of ADCs (e.g., enhanced permeability and retention effect) and tumor microenvironmental factors (e.g., proteolytic payload release). Our findings challenge the role of HER2 as a biomarker predictive of T-DXd response in pediatric cancers and support further biomarker-agnostic clinical development of T-DXd in desmoplastic small round cell tumor and pediatric renal tumors.

Large-scale combination drug screens are generally considered intractable due to the immense number of possible combinations. Existing approaches use ad hoc fixed experimental designs then train machine learning models to impute unobserved combinations. Here we propose BATCHIE, an orthogonal approach that conducts experiments dynamically in batches. BATCHIE uses information theory and probabilistic modeling to design each batch to be maximally informative based on the results of previous experiments. On retrospective experiments from previous large-scale screens, BATCHIE designs rapidly discover highly effective and synergistic combinations. In a prospective combination screen of a library of 206 drugs on a collection of pediatric cancer cell lines, the BATCHIE model accurately predicts unseen combinations and detects synergies after exploring only 4% of the 1.4M possible experiments. Further, the model identifies a panel of top combinations for Ewing sarcomas, which follow-up validation experiments confirm to be effective, including the rational and translatable top hit of PARP plus topoisomerase I inhibition. These results demonstrate that adaptive experiments can enable large-scale unbiased combination drug screens with a relatively small number of experiments. BATCHIE is open source and publicly available (https://github.com/tansey-lab/batchie).

INTRODUCTION:Omacetaxine, a semisynthetic form of Homoharringtonine (HHT), was approved for the treatment of Chronic Myeloid Leukemia (CML). Previously, we have published the synthesis of this natural alkaloid and three of its derivatives: Deoxyharringtonine (DHT), Deoxyhomoharringtonine (DHHT), and Bis(demethyl)-deoxyharringtonine (BDHT), and reported its refractory activity against the HL-60/RV+ cells over-expressing P-glycoprotein 1 (MDR1). METHODS:In this study, we have explored the extent of this resistance by first expanding the panel of established cell lines and using a panel of 21 leukemia patient-derived primary cells. RESULTS:Herein, we have reported consistent resistance to HTT of K562-derived cells and to mitoxantrone of MES-SA/MX2-derived cells; all of them have been found to overexpress MDR1, while we have found U87MG-ABCG2 and H69AR cells to be very sensitive to HTT. In contrast, DHT, DHHT, and BDHT seemingly overcame this resistance due to the changes made to the acyl chain of HTT, rendering the derivatives less susceptible to efflux. Surprisingly, the leukemia primary cells were very sensitive to HHT and its derivatives with low nanomolar potencies, followed by a new class of CDC7 kinase inhibitors, the anthracycline class of topoisomerase inhibitors, the DNA intercalator actinomycin-D, and the vinca alkaloid class of microtubule inhibitors. The mechanism of cell death induced by HTT and DHHT was found to be mediated via caspase 3 cleavage, leading to apoptosis. CONCLUSION:Taken together, our results confirm that HHT is a substrate for MDR1. It opens the door to a new opportunity to clinically evaluate HHT and its derivatives for the treatment of AML and other cancers.

Abstract Large-scale combination drug screens are largely considered intractable due to the immense number of possible combinations. Existing approaches use ad hoc fixed experimental designs then train machine learning models to impute novel combinations. We introduce BATCHIE, an orthogonal approach that adaptively conducts experiments in batches. BATCHIE uses information theory and probabilistic modeling to design each batch to be maximally informative based on the results of previous experiments. BATCHIE is fully modular, allowing any Bayesian probabilistic model to be used and any study constraints to be incorporated while maintaining optimality guarantees. Results: In retrospective simulations on public combination screens, BATCHIE saved 10s of thousands to 100s of thousands of experiments relative to non-adaptive baselines. We conducted a prospective study focusing on pediatric sarcomas with BATCHIE. Our study covered 16 cell lines spanning Ewing sarcoma (EWS), osteosarcoma, rhabdomyosarcoma, as well as non-sarcoma cancers and non-cancer lines. We used a drug library of 206 drugs at two doses. After 15 rounds of BATCHIE-driven data collection, we observed 54K unique combinations, covering 4% of the experimental landscape. On unobserved validation data, the BATCHIE model predictions were highly accurate (Pearson’s rho=0.91, p<10-30) and detected the rare (0.004%) combinations with significant synergy (AUC of ROC=0.85, p<10-5). We further investigated 10 combinations that BATCHIE predicted to have high therapeutic index (TI) for a broad selection of EWS lines, meaning a large differential effect between the predicted viabilities of the control lines and the target lines. We found that the TI scores for the top hits were significantly larger than the rest of the screen (p<10−50), with the median top hit TI score lying in the 98th percentile of observed TI scores. We further validated 6 of the top hits in an ex vivo study on 2 patient-derived EWS samples, again finding significantly large TI values (p<10-13), with the median ex vivo TI score lying in the 96th percentile of observed TI scores. The top hits also exhibited biologically plausible rationales including combining PARP inhibitors with topoisomerase 1 inhibitors and alkylating agents, despite our model utilizing no prior knowledge on the molecular targets of our drug library. Combining PARP inhibitors with topoisomerase 1 inhibitors comprise 3 of the 6 currently open phase II clinical combination trials for EWS. Citation Format: Christopher Tosh, Mauricio Tec, Jessica White, Jeffrey F. Quinn, Glorymar Ibanez Sanchez, Paul Calder, Andrew L. Kung, Filemon S. Dela Cruz, Wesley Tansey. BATCHIE: An active learning platform for scalable combination drug screens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 901.

10049 Background: Trastuzumab deruxtecan (T-DXd) is an anti-HER2 antibody-drug conjugate (ADC) linked to a topoisomerase inhibitor FDA-approved for several indications, including the treatment of HER2+ breast and gastric cancer. Although HER2 amplification is uncommon in pediatric cancers, recent demonstration of T-DXd efficacy in HER2-low breast cancer patients prompted us to examine the potential clinical relevance of T-DXd in pediatrics by evaluating HER2 expression and activity of T-DXd in preclinical pediatric solid tumor models. Methods: Cell viability was assessed in a panel of histologically diverse pediatric solid tumor cell lines and 2 HER2-amplified adult cancer cell lines treated with T-DXd, payload (DXd), and a control IgG-ADC. HER2 protein expression in patient-derived xenograft (PDX) tumors was evaluated by immunohistochemistry (IHC) using 5 clinically validated anti-HER2 antibodies. RNAseq was performed on clinical (n=290) and PDX (n=184) tumors to determine relative expression of ERBB2. In vivo activity of T-DXd was evaluated in osteosarcoma (OS, n=10), Wilms tumor (WT, n=1) and malignant rhabdoid tumor (MRT, n=1) PDX models and a desmoplastic small round cell tumor (DSRCT) cell line xenograft model. Differences in tumor volume and time to disease progression was assessed and compared across treatments and models. Responses were correlated with HER2 expression by IHC. Results: In vitro, HER2-amplified control cell lines demonstrated a >30-fold reduction of IC50 when comparing T-DXd to ADC control. In contrast, the ADC control IC50 was nearly identical to T-DXd across all pediatric cancer cell lines, consistent with the absence of HER2 amplification in these models. We observed focal and membranous staining of HER2 by IHC in PDXs but was quite variable across antibodies tested: WT 0-17% HER2+ cases (n=6), MRT 0-40% (n=5), DSRCT 0-33% (n=24), OS 0-7% (n=30). ERBB2 gene expression was highest in DSRCT followed by WT, OS and MRT. In vivo efficacy studies demonstrated complete and partial responses in OS, WT, and DSRCT and improved disease control rates (OS: 67%, p=0.006, Mann-Whitney; WT: 100%; DSRCT: 100%). However, T-DXd and ADC control demonstrated similar activity in all tumor types with no consistent correlation between in vivo response and HER2 expression. Consistent with these preclinical studies, 4 patients with progressive DSRCT were treated with T-DXd via compassionate/off-label access with signs of clinical and radiographic responses. Conclusions: T-DXd shows significant preclinical antitumor activity across multiple pediatric solid tumors but little correlation with HER2 expression suggesting a mechanism of action similar to the clinical activity observed in HER2-low breast cancer. Xenograft efficacy studies and preliminary clinical experience with T-DXd in DSRCT patients warrant formal clinical trial investigation in this largely incurable disease.

Supplementary figures and tables

Dengue is the most common mosquito-borne viral disease that in recent years has become a major international public health concern. Dengue is a tropical neglected disease with increasing global incidences, affecting millions of people worldwide, and without the availability of specific treatments to combat it. The identification of host-target genes essential for the virus life cycle, for which effective modulators may already exist, would provide an alternative path to a rapid drug development of the much needed antidengue agents. For this purpose, we performed the first genome-wide RNAi screen, combining two high-content readouts for dengue virus infection (DENV E infection intensity) and host cell toxicity (host cell stained nuclei), against an arrayed lentiviral-based short hairpin RNA library covering 16,000 genes with a redundancy of at least 5 hairpins per gene. The screen identified 1924 gene candidates in total; of which, 1730 gene candidates abrogated dengue infection, whereas 194 gene candidates were found to enhance its infectivity in HEK293 cells. A first pass clustering analysis of hits revealed a well-orchestrated gene-network dependency on host cell homeostasis and physiology triggering distinct cellular pathways for infectivity, replication, trafficking, and egress; a second analysis revealed a comprehensive gene signature of 331 genes common to hits identified in 28 published RNAi host-viral interaction screens. Taken together, our findings provide novel antiviral molecular targets with the potential for drug discovery and development.

Background: Outcomes for pediatric patients with relapsed or refractory acute myelogenous leukemia (AML) are poor, with <30% overall survival in recurrent settings. Additionally, patients harboring high-risk molecular features, including KMT2A/MLL-rearrangements or GLIS2 fusion genes, experience poor outcomes. LSD1 inhibition has been evaluated as a potential therapeutic approach in adult patients with AML, but its role in pediatric AML remains limited. In this study, we characterize the anti-leukemic effect of an LSD1 inhibitor, IMG-7289, across a panel of pediatric AML models in vitro and in vivo and evaluate its effect on leukemia initiating cells (LICs). Methods: Dose-response effect of IMG-7289 on cell viability was assessed across 11 pediatric leukemia cell lines. Induction of apoptosis and cell cycle arrest was assessed by flow cytometric analysis of Annexin V and 7-AAD respectively. Flow cytometry was also used to analyze a panel of cell surface markers to assess for differentiation effect. Pediatric AML patient-derived xenograft (PDX) mouse models, including an acute megakaryoblastic leukemia (AMKL) model and a KMT2A-rearranged (KMT2A-r) AML model, established by implanting AML cells into NSG-S mice, were used for in vivo validation studies and limiting dilution secondary transplantation studies. PDX models were treated with IMG-7289 (25 mg/kg PO) or Vehicle daily for 21 days (n=6/arm). Engraftment was determined using flow cytometry to assess expression of human CD45 (hCD45) in the bone marrow. Leukemia initiating cell (LIC) frequency was evaluated by transplanting serial dilutions (2x105, 105, 104, 103, and 102 cells) of bone marrow cells derived from Vehicle- or IMG-7289-treated PDX models into untreated donor mice. The LIC frequency was estimated using the Extreme Limiting Dilution Analysis (ELDA) software (Walter and Eliza Hall Bioinformatics Institute of Medical Research), and log-rank analysis was used to compare event-free survival (EFS), defined as time from secondary transplant to engraftment. Results: Seven pediatric AML cell lines demonstrated sensitivity to IMG-7289, including 2 AMKL cell lines, M07e (IC50: 0.03 μM ± 0.01) and CMK (IC50: 0.065 μM ± 0.003), and 2 KMT2A-r AML cell lines, MV4;11 (IC50: 0.007 μM ± 0.001) and MOLM14. Treatment with IMG-7289 leads to induction of apoptosis (DMSO 8.8% vs IMG-7289 30.3%, P=0.0005) and cell-cycle arrest (G0/G1 phase populations: DMSO 65.5% vs IMG-7289 74.7%, P<0.0001) in MV4;11 cells. To investigate the effect of IMG-7289 on induction of cell differentiation, cell surface marker changes were evaluated over time in IMG-7289-treated AML. MV4;11 and M07e cells treated over 25 days showed increased expression of the monocytic cell surface markers CD14 and CD86 compared to DMSO control. Additionally, decreased expression of megakaryocytic markers CD41 and CD42b were observed in IMG-7289-treated M07e cells. To evaluate the in vivo activity of IMG-7289, a KMT2A-r AML PDX model was treated with IMG-7289 for 21 days. We observed a significant reduction in hCD45+ cells in IMG-7289-treated mice (27.5% hCD45+) vs Vehicle-treated mice (76% hCD45+, P=0.0012). To evaluate effects of IMG-7289 on the LIC population, serial transplantation of cells derived from Vehicle-treated or IMG-7289-treated AMKL PDX model was performed. Assessment of leukemia engraftment (flow cytometric analysis for hCD45+ cells in mouse bone marrow) at 20 weeks post-secondary transplant demonstrated an LIC frequency of 1 in 4.9x104 in IMG-7289-treated mice representing a ~500-fold reduction in LIC number compared to Vehicle control. Furthermore, a significant improvement in EFS was observed in IMG-7289-treated mice (median survival = 33 days) compared to Vehicle control (median survival = 76 days, P=0.0009, log-rank). Conclusions: In conclusion, treatment with IMG-7289 results in decreased viability, as demonstrated by induction of apoptosis and cell cycle arrest, as well as differentiation induction in pediatric AML cell lines. The in vitro and in vivo activity observed in pediatric AML is also observed in models with high-risk phenotypes (KMT2A-r AML and AMKL). Activity of LSD1 inhibition on the LIC population may represent a promising strategy to mitigate relapse or refractory disease.

Limited clinical data are available regarding the utility of multikinase inhibition in neuroblastoma. Repotrectinib (TPX-0005) is a multikinase inhibitor that targets ALK, TRK, JAK2/STAT, and Src/FAK, which have all been implicated in the pathogenesis of neuroblastoma. We evaluated the preclinical activity of repotrectinib monotherapy and in combination with chemotherapy as a potential therapeutic approach for relapsed/refractory neuroblastoma. In vitro sensitivity to repotrectinib, ensartinib, and cytotoxic chemotherapy was evaluated in neuroblastoma cell lines. In vivo antitumor effect of repotrectinib monotherapy, and in combination with chemotherapy, was evaluated using a genotypically diverse cohort of patient-derived xenograft (PDX) models of neuroblastoma. Repotrectinib had comparable cytotoxic activity across cell lines irrespective of ALK mutational status. Combination with chemotherapy demonstrated increased antiproliferative activity across several cell lines. Repotrectinib monotherapy had notable antitumor activity and prolonged event-free survival compared with vehicle and ensartinib in PDX models (P < 0.05). Repotrectinib plus chemotherapy was superior to chemotherapy alone in ALK-mutant and ALK wild-type PDX models. These results demonstrate that repotrectinib has antitumor activity in genotypically diverse neuroblastoma models, and that combination of a multikinase inhibitor with chemotherapy may be a promising treatment paradigm for translation to the clinic.

Background: Acute myeloid leukemia (AML) is a very aggressive bone marrow malignancy which carries a poor prognosis despite intensive chemotherapy. The treatment of relapsed and refractory AML remains suboptimal. Although more novel therapies are being introduced for AML, there is a limitation of appropriate, predictive, preclinical models available to identify and test novel therapies. In-vitro drug sensitivity testing of patient-derived AML cells is increasingly being used to facilitate treatment options. However, these tests are often done in suboptimal conditions with difficulty in the interpretation of results. Accumulating evidence has shown that the long-term, in-vitro, survival of primary AML cells can be supported with stromal co-culture, which would also take into account the influence of the surrounding tumor microenvironment. However, there has been no direct comparison of a stromal co-culture method to non-stromal growth method of primary AML cells. This is the first comprehensive direct comparison of the proliferation and immunophenotypes of primary AML cells under two different stromal conditions and cytokines, the results of which would not only highlight the importance of stromal cells to chemotherapy resistance but also lay the groundwork for the feasibility and importance of comparing it to non-stromal drug sensitivity testing.

Compound optical interference remains an inherent problem in chemical screening and has been well documented for biochemical assays and less so for automated microscopy-based assays. It has also been the assumption that the latter should not suffer from such interference because of the washing steps involved in the process, thus eliminating the residual nonspecific compound effects. Instead, these compounds may have no relevance to the actual target, and as such, compound optical interference contributes to a number of false-positives, resulting in a high attrition rate during subsequent follow-up studies. In this report, we analyze the outcome of a high-content screen using enhanced green fluorescent protein as a reporter in a gain-of-function cell-based assay in search of modulators of the micro RNA (miRNA) biogenesis pathway. Using a previously validated image-based biosensor, we screened a diverse library collection of ~315,000 compounds covering natural and synthetic derivatives in which 1130 positives were identified to enhance green fluorescence expression. Lateral confirmation and dose-response studies revealed that all of these compounds were the result of optical interference and not specific inhibition of miRNA biogenesis. Here, we highlight the chemical classes that are susceptible to compound optical interference and discuss their implications in automated microscopy-based assays.

e18535 Background: To individualize therapy for relapsed/refractory AML patients, optimal in-vitro culture conditions to support primary leukemic cells are essential for drug sensitivity testing. Our lab has validated a high throughput chemosensitivity assay with primary AML cells maintained by growth factors (cytokines); however, growth factors have not been shown to support long-term assays of primary AML cells. Stromal cells of the tumor microenvironment are crucial to maintain normal hematopoiesis and leukemic cells and have been shown to support long term in-vitro expansion of primary AML cells. However, there is little information characterizing these growth conditions. The aim of this study was to compare long-term proliferation and phenotypes of primary AML cells with growth factors or stromal support to best determine their utility as a platform for drug sensitivity testing in functional assays. Methods: Patient-derived AML cells were cultured in 96-well plates in: 1) cell culture medium only 2) Human HS5 or HS27 stromal cells 3) HS5 or HS27 stroma-conditioned media or 4) cytokine cocktail. Viability readout by Guava ViaCount and leukemic cell surface phenotypes by fluorescently-conjugated antibodies were performed weekly over 3 weeks. Results: Primary AML cells cultured with only cytokines maintained proliferation at 3 weeks. In comparison, AML cells cultured in HS5 stroma-conditioned medium also maintained proliferation at a similar rate at 3 weeks, while co-culture with HS5 stromal cells demonstrated significantly higher proliferation. Leukemic immunophenotypes were maintained for all growth conditions over 3 weeks. Conclusions: Contrary to known data, primary AML cells with cytokines continued to expand at 3 weeks, at a similar rate to HS5 stroma-conditioned medium, a finding that has not been reported. Consistent with previous studies, we confirmed that stromal cells such as HS5 can provide long-term in-vitro expansion of primary AML cells, which cannot be substituted by stroma-conditioned medium. The ability to maintain long-term expansion of primary AML cells by both cytokines and stromal cells sets up a platform for a direct comparison of high throughput drug sensitivity testing under these growth conditions.

Background: Acute myeloid leukemia (AML) is a very aggressive hematological malignancy that carries an overall poor prognosis. High throughput screening of primary (patient-derived) leukemic cells is becoming increasingly valuable as a preclinical tool to help individualize the treatment of relapsed/refractory AML patients. However, the necessity of robust in-vitro culture conditions has often limited the optimization of high throughput drug sensitivity testing. Our lab has previously validated a high throughput assay in 384-well format that utilizes primary leukemic cells maintained in a cocktail of cytokines tested for chemosensitivity and resistance against anti-leukemic agents (Shum et al. Clinical Lymphoma, Myeloma and Leukemia 2013). However, cytokines have not been shown to support long-term proliferation of primary AML cells; nor does it assess drug candidates within the appropriate tumor microenvironment. Stromal cells of the bone marrow microenvironment, however, have been reported to support not only the growth and maintenance of normal hematopoiesis, but also the long-term, in-vitro growth of leukemic cells, while also influencing their response to chemotherapy agents. A comprehensive comparison of primary AML cell growth under our validated cytokine assay and stromal conditions has not previously been undertaken. In this study, we compared the long-term proliferation and phenotypes of primary AML cells in high-throughput format under different stromal conditions against cytokines only. Results from this study would help optimize parameters for a high-throughput drug sensitivity testing comparing the differential response to anti-leukemic agents under cytokine versus stromal conditions.

A universal process in experimental biology is the use of engineered cells; more often, stably or transiently transfected cells are generated for the purpose. Therefore, it is important that cell health assessment is conducted to check for stress mediated by induction of heat shock proteins (Hsps). For this purpose, we have developed an integrated platform that would enable a direct assessment of transfection efficiency (TE) combined with cellular toxicity and stress response. We make use of automated microscopy and high content analysis to extract from the same well a multiplexed readout to assess and determine optimal chemical transfection conditions. As a proof of concept, we investigated seven commercial reagents, in a matrix of dose and time, to study transfection of an EGFP DNA plasmid into HeLa cells and their consequences on health and fitness; where we scored for cellular proliferation, EGFP positive cells, and induction of Hsp10 and Hsp70 as makers of stress responses. FuGENE HD emerged as the most optimal reagent with no apparent side effects suitable for performing microtiter based miniaturized transfection for both chemical and RNAi screening. In summary, we report on a high content assay method to assess cellular overall fitness upon chemical transfection.

Memorial Sloan Kettering Cancer Center (MSKCC) has implemented the creation of a full service state-of-the-art High-throughput Screening Core Facility (HTSCF) equipped with modern robotics and custom-built screening data management resources to rapidly store and query chemical and RNAi screening data outputs. The mission of the facility is to provide oncology clinicians and researchers alike with access to cost-effective HTS solutions for both chemical and RNAi screening, with an ultimate goal of novel target identification and drug discovery. HTSCF was established in 2003 to support the institution's commitment to growth in molecular pharmacology and in the realm of therapeutic agents to fight chronic diseases such as cancer. This endeavor required broad range of expertise in technology development to establish robust and innovative assays, large collections of diverse chemical and RNAi duplexes to probe specific cellular events, sophisticated compound and data handling capabilities, and a profound knowledge in assay development, hit validation, and characterization. Our goal has been to strive for constant innovation, and we strongly believe in shifting the paradigm from traditional drug discovery towards translational research now, making allowance for unmet clinical needs in patients. Our efforts towards repurposing FDA-approved drugs fructified when digoxin, identified through primary HTS, was administered in the clinic for treatment of stage Vb retinoblastoma. In summary, the overall aim of our facility is to identify novel chemical probes, to study cellular processes relevant to investigator's research interest in chemical biology and functional genomics, and to be instrumental in accelerating the process of drug discovery in academia.