Division Of Cancer Treatment Research Articles

Targeting AMP kinase in melanoma: A phase I trial of phenformin with dabrafenib/trametinib.

9536 Background: AMP-activated protein kinase (AMPK) can phosphorylate BRAF and block its activation of MEK. AMPK is activated by biguanides such as metformin and phenformin (phen). Preclinical data indicated that phen can enhance the effects of BRAF inhibitors (BRAFi) in sensitive and BRAFi-resistant melanoma (mel) cell lines whereas metformin does not. Phen also can decrease myeloid-derived suppressor cells (MDSCs) enhancing immune reactivity against mel. For these reasons, we designed a phase I trial combining phen with dabrafenib/trametinib (dab/tram) in patients (pts) with metastatic BRAF V600-mutated mel. Methods: Bulk phen was provided by the Division of Cancer Treatment and Diagnosis of the National Cancer Institute under a materials transfer agreement. Phen was encapsulated at the Pharmaceutical Product Facility at MSKCC under cGMP procedures and used under an IND held by MSKCC. Eligible pts had metastatic BRAF V600-mutated mel. For the dose-escalation phase, prior BRAFi therapy was allowed. A 3+3 dose escalation design was used starting at a phen dose of 50 mg bid and standard dosing of dab/tram (150 mg bid/2 mg qd). Subsequent planned dose levels were 100 mg bid, 200 mg bid, and 300 mg bid with de-escalation to intermediate doses for toxicity. Plasma PK samples were collected and assayed by MRI Global Inc using a validated, GLP-compliant, HPLC-MS/MS assay. PBMC were collected to measure circulating MDSCs. At the recommended phase 2 dose (RP2D), we planned to treat 10 BRAFi-naïve pts. Results: We accrued 18 patients from March 2017 through December 2019. Dose-escalation to 200 mg bid was not tolerable nor was 150 mg bid. Therefore, we determined 100 mg bid to be the RP2D and accrued 2 patients on the dose expansion cohort before further accrual became impossible due to the COVID-19 pandemic. The most common toxicities were gastrointestinal – nausea, vomiting, anorexia, and transaminase and alkaline phosphatase elevation – accounting for 9 DLTs. Two patients were hospitalized due to lactic acidosis. There were also constitutional/inflammatory and skin/mucosal adverse events thought to be related to dab/tram. Weight loss was seen in most patients (median 3.7% at week 8) thought to be related to phen. There were 8 PRs and 2 CRs. Responses were observed in 2/7 pts who had received prior therapy with BRAFi. One CR lasted > 1 year. There were 8/11 (73%) responders among the BRAFi-naïve pts. At the RP2D of 100 mg bid, day 8 median phen plasma level was 73.4 ng/ml (range 32.8-253.2). At the the 50 mg bid dose level, it was 40.8 ng/ml (range 29.7-110.2). MDSC could be analyzed from the 150 mg bid and 200 mg bid dose levels (N = 7 pts). In all but 1 pt, MDSCs decreased while on phen. Conclusions: We observed 10/18 (55.5%) total responses with 2/7 among previously treated pts. Circulating MDSCs decreased on treatment. Phen at 100 mg bid can be combined safely with dab/tram and should be explored in future randomized phase II trials. Clinical trial information: NCT03026517 .

Abstract PS18-46: Inhibition of breast cancer stem cells in 2- and 3-dimensional culture by novel salinomycin analogs

Abstract Breast cancer remains one of the leading cancers among women and an estimated 90% of breast cancer deaths are due to metastasis. Cancer stem cells (CSCs) are a sub-population of cancer cells which are responsible for its initiation, progression and metastasis. CSCs are resistant to conventional chemo- and radio-therapies and therefore therapeutic strategies targeting CSCs hold great potential for novel advances in cancer treatment. Salinomycin (SAL) is a naturally occurring polyether ionophore antibiotic which was shown to effectively target breast CSCs. A library of 17 novel SAL analogs was synthesized and screened to identify compounds with improved selectivity against breast cancer stem cells. SAL analogs were either single modified esters or amides in the C1 position or double-modified C20-oxo derivatives. Eight single- and two double-modified analogs were more potent (IC50 range of 1.13 ± 0.19 to 3.93 ± 0.39 µM) towards the breast cancer cell line MDA-MB-231 compared to parent SAL (IC50 of 4.90 ± 1.60 µM). These analogs induced DNA fragmentation suggestive of apoptotic cell death. Compounds 2 (butyl ester analog of SAL) and 17 (double-modified C20-oxosalinomycin with benzhydroxamic acid) have been chosen for follow-up screening due to their improved activity and selectivity versus parent SAL. This included clonogenic assays to assess the ability of the compounds to affect cell renewal, and wound healing assays to assess cell migratory properties. In both assays, compound 17 showed superior properties over SAL. Furthermore, analog 17 showed improved targeting of breast CSCs in both cell monolayer and organoid culture as assessed in assays measuring the CD44+/CD24- stem cell sub-population. Analogs and parent compound were further studied examining (ADP-ribose) polymerase (PARP) cleavage and Bcl-2 levels by immunoblotting. All three compounds induced loss of 116 kDa PARP expression within 48h, with the highest effect induced by analog 17. In addition, treatment with compound 17 caused a decrease in Bcl-2 expression as early as 24 h. Select analogs were next screened against the NCI-60 Human Tumor Cell Line Panel. The described above double-modified analog was found to be more potent than SAL towards all 6 breast cancer cell lines in the panel as well as other tumor types. The present findings highlight the therapeutic potential of SAL analogs towards breast stem cells and support further research and clinical development of these compounds. Funding: The present study was funded by grants (to AM and TCC) from the Arkansas Breast Cancer Research Program. Testing was performed by the Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, http://dtp.cancer.gov. Citation Format: Alicja Joanna Urbaniak, Megan R. Reed, Michał Antoszczak, Michał Sulik, Adam Huczyński, Robert L. Eoff, Melanie C. MacNicol, Timothy C. Chambers, Angus M. MacNicol. Inhibition of breast cancer stem cells in 2- and 3-dimensional culture by novel salinomycin analogs [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-46.

Abstract PO-092: Expanded access trial of tocilizumab in COVID19+ hospitalized cancer patients

Abstract Introduction: Emerging data from the COVID-19 pandemic suggest that an IL6-mediated cytokine release syndrome (CRS) may contribute to disease severity. Tocilizumab (Toci), an anti-IL6 receptor antibody, is FDA approved for the treatment of chimeric antigen receptor T cell-associated CRS for patients (pts) as young as 2 years of age. Phase 3 randomized testing of toci in pts with severe COVID-19 disease is ongoing. Cancer pts infected with SARS-CoV-2 are at risk for life-threatening or fatal complications. Outcomes are particularly dismal for those with hematologic malignancies or lung cancer. Due to disease and treatment-related complications, cancer patients may not be eligible for enrollment on COVID-19 directed studies, including the aforementioned randomized phase 3 study of toci. Furthermore, access to clinical trials is frequently restricted to those who are adults; however, children with cancer may also be predisposed to developing more severe complications from COVID-19. Improving access to clinical trial enrollment for vulnerable, minority, and underserved populations at increased risk of complications from COVID-19 is imperative. Methods: We developed a multicenter, single-arm, open-label, expanded-access treatment study to provide toci to cancer pts with COVID-19. Coordinated through the National Cancer Institute, Cancer Therapy Evaluation Program, and the Division of Cancer Treatment and Diagnosis, this expanded-access trial is being conducted to provide clinical trial access to this potentially beneficial therapy in cancer pts and to collect data on clinical outcomes. The primary objective is to enhance access to toci for cancer patients who cannot participate in the randomized phase 3 toci trial with a specific emphasis on enrollment of those who belong to high-risk and minority populations and children. Hospitalized cancer subjects > 2 years of age with COVID-19 respiratory compromise may be eligible to receive toci. Treatment will allow for up to two doses of toci and will also evaluate the efficacy of a lower dose of toci in those who are not intubated. Concurrent receipt of investigational cancer and/or antiviral therapy and other supportive therapies is permitted. Exploratory objectives will include analysis of inflammatory cytokines, monitoring of SARS-CoV-2 viral load, and determination of pharmacokinetics of toci to facilitate exposure-response analysis. Results: This trial was officially activated on May 28, 2020, and is being opened at multiple sites across the United States from the National Cancer Institute’s Community Oncology Research Program, the National Clinical Trials Network, and the Experimental Therapeutics Clinical Trials Network. Updates will be presented at the AACR Virtual Meeting: COVID-19 and Cancer. Conclusions: Targeting an unmet need, this trial provides a prospective mechanism for tocilizumab access and clinical outcome collection in cancer patients with severe COVID-19 disease. Citation Format: Nirali N. Shah, Percy Ivy, Rebecca Enos, Matthew J. Boron, Rodney Howells, Boris Freidlin, Carmen Allegra, Margaret M. Mooney, Worta McCaskill-Stevens, James H. Doroshow, Richard F. Little. Expanded access trial of tocilizumab in COVID19+ hospitalized cancer patients [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2020 Jul 20-22. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(18_Suppl):Abstract nr PO-092.

Abstract 3913: Evaluation of patient-derived cell lines and cancer organoids for the prediction of drug responses in patient-derived xenograft models

Abstract Cancer organoids are heterogeneous 3D cellular clusters with complexities that mimic some characteristics of tumors in situ. Thus, assays performed with cancer organoids might enable better predictions of in vivo drug responses than those performed with cell monolayers. The National Cancer Institute (NCI) is developing a national repository of Patient-Derived (PD) models comprised of clinically annotated and molecularly characterized PD xenografts (PDXs), PD tumor cell lines (PDCs), and PD cancer organoids (PDOrgs) (https://pdmr.cancer.gov/). We evaluated the therapeutic activity of a panel of FDA-approved and investigational anticancer agents, including carboplatin, gemcitabine, paclitaxel, SN38, 5-FU, adavosertib, erlotinib, trametinib, and vemurafenib, against a cohort of PDCs, PDOrgs, and PDXs from solid tumors including colon, gastroesophageal, head and neck, NSCLC, pancreatic, bladder, and uterine cancers. Our goal was to investigate whether drug sensitivities determined using PDCs and PDOrgs correlate with responses observed in the matching PDXs. Cultures were exposed to anticancer agents at concentrations ranging from 1 pM to 100 µM for periods of 4 or 6 days. The data indicated that the GI50 values for PDOrgs were in overall agreement with in vivo PDX drug responses measured as relative median to event free survival (RMEFS), where an event is the median time (days) from treatment initiation to tumor volume quadrupling, calculated as median time to tumor volume quadrupling for treated animals/median time to tumor volume quadrupling for control animals. For both paclitaxel and trametinib, responses in PDOrgs, from most sensitive to most resistant, were similar to the corresponding PDXs. Drug sensitivities determined in PDC monolayers were less clearly related to in vivo PDX responses; particularly for PDCs treated with carboplatin, gemcitabine, and SN-38. This work is part of a larger effort to provide a rigorous comparison between fully characterized and annotated PDCs-PDOrgs-PDXs to assess the value of different in vitro model systems for the prediction of PDX drug responses. This research was supported [in part] by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Petreena Campbell, Curtis Hose, Lara El Touny, Erik Harris, John Connelly, Carrie Bonomi, Kelly Dougherty, Savanna Styers, Abigail Walke, Jenna Moyer, Mariah Baldwin, Anna Wade, Michael Mullendore, Kaitlyn Arthur, Matthew Murphy, Kevin Plater, Marion Gibson, Joseph Geraghty, Michelle Gottholm-Ahalt, Tara Grinnage-Pulley, Tiffanie Chase, John Carter, Howard Stotler, Debbie Trail, Luke Stockwin, Dianne Newton, Yvonne Evrard, Melinda Hollingshead, Ralph E. Parchment, Nathan P. Coussens, Beverly A. Teicher, James H. Doroshow, Annamaria Rapisarda. Evaluation of patient-derived cell lines and cancer organoids for the prediction of drug responses in patient-derived xenograft models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3913.

Oncolytic virus pelareorep plus carfilzomib phase I trial in carfilzomib-refractory patients (NCI 9603): Responses with cytokine storm.

8535 Background: Viral oncolytic therapy with intravenous Pelareorep, the infusible form of reovirus, is supported by preclinical data indicating that its antitumor activity results from direct cytolysis and an immune response against infected MM cells. Our preclinical data has shown that monocytes serve as carriers of reovirus from the peripheral blood to the bone marrow (Dona et al, ASH, 2019). In this abstract we present patients treated to date that were Carfilzomib refractory at enrollment. Methods: Pelareorep (P), Carfilzomib (K), and dexamethasone (d) were all infused on days 1, 2, 8, 9, 15 and 16 of a 28-day cycle. Patients were pretreated with dexamethasone 20 mg intravenously, then Carfilzomib 20 mg/m2 on days 1 and 2 of cycle 1, and 56 mg/m2 thereafter. Pelareorep dose levels were at 3x1010, 4.5*1010, and planned 9*1010 median tissue culture infectious dose (TCID50). Results: All 6 evaluable patients showed reovirus infection in the post-treatment marrow aspirates cycle 1 day 9. Despite all patients having Carfilzomib-refractory disease, 2 patients achieved partial responses, two patients with short duration stable disease but few side effects, and 2 patients with PD. Of the two responders, one patient developed fever and grade 4 thrombocytopenia during cycle 1 and withdrew mid cycle. The other patient with a PR developed a cytokine storm - this patient presented cycle 1 day 3 hypoxemic with pneumonia, systolic failure (LVEF 69%-- > 26%), with biochemical evidence of hemophagocytic syndrome with elevated IL-2R, IL-6, and ferritin > 25K. This patient responded to tocilizumab, but ultimately died two weeks later with sepsis. Autopsy revealed pneumonia, no areas of reovirus infection other than in myeloma cells, and reovirus productive infection in 5-35% of myeloma cells (i.e. capsid protein). Conclusions: While infection of myeloma cells was seen in all evaluable patients on cycle 1 day 9, those with a clinical response demonstrated concomitant CD8 & NK cell recruitment, PD L1 upregulation, activated caspase-3 expression, increased viral protein production within the myeloma cells, and these patients demonstrated mild to severe signs and symptoms consistent with secondary HLH. This is the first report of cytokine storm after oncolytic virus in a patient with a blood cancer, a syndrome thought to be related to T-cell activation from the combination treatment. This trial was supported by the NCI Division of Cancer Treatment & Diagnosis Cancer Therapy Evaluation Program (CTEP). Clinical trial information: NCT02101944 .

Novel Salinomycin Analogs Show Improved Selectivity Towards Breast Cancer Stem Cells

Breast cancer tumor metastasis is the leading cause of mortality among women. Cancer stem cells (CSCs) are the tumor‐initiating cells which drive initiation, progression, metastasis, and tumor reoccurrence. Their resistance to conventional chemo‐ and radiotherapies and their ability to survive such treatment enables tumor reestablishment. Thus, therapeutic strategies targeting CSCs hold great potential for novel advances in cancer treatment. Salinomycin (SAL) is a naturally occurring polyether ionophore antibiotic with anti‐cancer activity towards various types of tumor cells, but its toxicity towards normal cells limits broad application. A library of 21 novel SAL analogs was screened to identify compounds with improved selectivity especially against breast cancer stem cells. SAL analogs were either single modified with ester or amide in the C1 position or double‐modified C20‐oxo derivatives. Eight single‐ and two double‐modified derivatives were more potent (IC50 range of 1.13 ± 0.19 to 3.93 ± 0.39 μM) towards the breast cancer cell line MDA‐MB‐231 compared to SAL (IC50 of 5.81 ± 1.50). These derivatives induced DNA fragmentation suggestive of apoptotic cell death. Further, most of these derivatives had improved selectivity toward MDA‐MB‐231 cells compared to SAL when also tested against normal breast epithelial MCF10A cells. In addition, a butyl ester derivative of SAL, chosen based on increased potency and selectivity versus SAL, significantly reduced the colony‐forming efficiency of MDA‐MB‐231 cells in association with selective loss of the CD44+/CD24−/low stem‐cell‐like subpopulation. Select derivatives were next screened against the NCI‐60 Human Tumor Cell Line Panel. A double‐modified analog of C20‐oxo‐SAL with hydroxamic acid was found to be more potent than SAL towards all 6 breast cancer cell lines in the panel as well as other tumor types especially leukemia. Studies are underway to test the analogs in three dimensional culture using breast cancer organoids derived from both cell lines and patients. The present findings highlight the therapeutic potential of SAL analogs towards breast cancer and breast cancer stem cells and support further research and clinical development of these compounds.Support or Funding InformationThe present study was funded by grants (to AMM and TCC) from the Arkansas Breast Cancer Research Program. Testing was performed by the Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, http://dtp.cancer.gov.

Abstract 270: Temozolomide resistant MGMT negative/MMR proficient cancer cells rely on ATR signaling and homologous recombination for DNA repair and survival

Abstract MGMT loss is used to predict response to temozolomide (TMZ), however, some patients do not benefit, suggesting that additional criteria for selection remain to be identified. Using patient derived and well-established cancer lines cancer lines we observed a 100% association between mismatched repair (MMR) defects and TMZ resistance; however, only 16% of MGMT-/MMR+ lines were sensitive to TMZ at clinically relevant concentrations. Activation of DNA damage and repair signaling was noted in both sensitive and resistant bladder cancer cell lines in response to TMZ but protracted DNA damage, accompanied by persistent G2/M arrest, was observed only in the sensitive models. ATR inhibition sensitized MGMT-/MMR proficient resistant models to TMZ resulting in measurable DNA damage, prolonged G2/M arrest and persistent growth inhibition: an effect that was mediated by Chk1. Homologous recombination and repair has been shown to play an important role in repairing TMZ-induced DNA damage. We demonstrate that ATR inhibition abrogated homologous recombination (HR) activity in a MGMT-/MMR proficient TMZ resistant bladder model and that a TMZ sensitive bladder model had minimal HR (assessed by pDR-GFP/SceI assay). Moreover, HR -deficiency (mediated by BRCA1 defects or RAD51 inhibition) conferred sensitivity to TMZ in combination with an ATR inhibitor. Our in vitro data suggest that a MGMT-/MMR+/HR-Deficiency profile identifies models that respond to TMZ/ATR inhibitor combination. Indeed, one Patient-derived Xenograft (PDX) model (available in the Patient Derived Models Repository), selected by this criterion (MGMT-/MMR+ and LOH>40%) showed delayed tumor growth and increased survival in the TMZ-VX970 combination arm, compared to single agents. Overall, our data suggest that MGMT-/MMR+ cells rely on ATR-dependent signaling for repair of TMZ-induced DSBs and that HR defects should be evaluated as criteria for selecting patients that will benefit from an ATR inhibitor/TMZ combination regimen. Funded by NCI Contract No. HHSN261200800001E. This research was supported, in part, by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Citation Format: Lara El Touny, Erik Harris, Curtis Hose, John Connelly, Diana Vengsarkar, Anne Monks, Ralph E. Parchment, James H. Doroshow, Beverly A. Teicher, Annamaria Rapisarda. Temozolomide resistant MGMT negative/MMR proficient cancer cells rely on ATR signaling and homologous recombination for DNA repair and survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 270.

Circulating Tumor DNA Assays in Clinical Cancer Research.

The importance of circulating free DNA (cfDNA) in cancer clinical research was recognized in 1994 when a mutated RAS gene fragment was detected in a patient's blood sample. Up to 1% of the total circulating DNA in patients with cancer is circulating tumor DNA (ctDNA) that originates from tumor cells. As ctDNA is rapidly cleared from the blood stream and can be obtained by minimally invasive methods, it can be used as a dynamic cancer biomarker for cancer early detection, diagnosis, and treatment monitoring. Despite the potential for clinical use, few ctDNA assays have been cleared or approved by the US Food and Drug Administration. As tools for clinical and translational research, current ctDNA assays face some challenges, and more research is needed to advance use of these assays. On September 29-30, 2016, the Division of Cancer Treatment and Diagnosis at the National Cancer Institute convened a workshop entitled "Circulating Tumor DNA Assays in Clinical Cancer Research" to garner input from industry experts, academia, and government research and regulatory agencies to understand and promote the translation of ctDNA assays to clinical research, with potential to advance to use in clinical practice. This Commentary presents the topics of the workshop covered in the presentations and points made in the discussions that followed: 1) background on ctDNA, 2) potential clinical utility of ctDNA assays, 3) assay technology, 4) assay clinical and analytical validation, and 5) industry perspectives. Additional relevant information that has come to light since the workshop has been included.

Abstract A116: Temozolomide-resistant MGMT/MSI double-negative cancer cells rely on ATR signaling and homologous recombination for DNA repair and survival

Abstract Although loss of MGMT expression is used as a predictive biomarker for response to temozolomide (TMZ), some patients do not benefit from treatment, suggesting that additional criteria for patient selection remain to be identified. We used a panel of newly developed patient derived models and well-characterized cancer lines to evaluate potential determinants of TMZ response and identify strategies to overcome TMZ resistance in MGMT negative cancers. There was 100% association between microsatellite instability (MSI) and TMZ resistance in the lines tested. Moreover, only a fraction of MGMT/MSI double-negative lines were sensitive to TMZ at clinically relevant concentrations (37.6µM). TMZ-induced DNA damage and repair (DDR) were compared in a bladder line sensitive to TMZ (BLX1, IC50 11.4µM) and a resistant large neuroendocrine cancer line (LNX1, IC50>80 µM). We observed DDR activation in both models, but only BLX1 had measurable DNA damage (PARP cleavage and γH2AX accumulation, starting 24 h post treatment). An extended G2/M arrest occurred in BLX1 but not in LNX1, suggesting that LNX1 efficiently repaired TMZ-induced damage. ATR inhibition (VE821, VX970 or AZD6738), but not ATM inhibition, in LNX1 cells increased response to TMZ (IC50 27.5µM), PARP cleavage, γH2AX accumulation, and protracted G2/M arrest. BLX1 had minimal homologous recombination (HR) capacity (measured by pDR-GFP/SceI assay) compared to LNX1; however, ATR inhibition abrogated HR activity in LNX1 cells, suggesting HR importance in repair of TMZ-induced damage. To assess whether HR defects sensitize cells to TMZ, we used MGMT positive BRCA1 null cells (UWB1.289) and their BRCA1 reconstituted counterpart line. BRCA1 status did not affect TMZ response in the presence of an MGMT inhibitor (TMZ IC50>80µM); however, BRCA1 null cells were sensitive to TMZ in combination with an ATR inhibitor (VE821 0.3µM, TMZ IC50 10µM). BRCA1 reconstituted cells were sensitized to TMZ+VE821 using a Rad51 inhibitor (B02 2.5µM, TMZ IC50 22.5µM), suggesting that when HR is compromised, cells rely on ATR signaling for repair of TMZ-induced damage. Our data suggest that MGMT/MSI double-negative cells rely on ATR-dependent signaling for repair of TMZ-induced DSBs and that HR defects could be used as additional selection criteria for TMZ+ATRi treatment. Funded by NCI Contract No. HHSN261200800001E. This research was supported, in part, by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Citation Format: Lara H. El Touny, Erik Harris, Curtis Hose, John Connelly, Anne Monks, Dianne Newton, Luke Stockwin, Melinda Hollingshead, Ralph Parchment, James H. Doroshow, Beverly A. Teicher, Annamaria Rapisarda. Temozolomide-resistant MGMT/MSI double-negative cancer cells rely on ATR signaling and homologous recombination for DNA repair and survival [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A116.

Abstract A117: Modeling ATM loss of function via CRISPR-Cas9 as a predictive tool for therapeutic responses in cancer cells

Abstract ATM is a master regulator of the DNA damage response (DDR) elicited by double-strand breaks (DSB). After DNA DSB, ATM controls cell fate via cell cycle arrest, apoptosis, and DNA damage repair. Numerous tumor types have ATM gene loss-of-function mutations, resulting in genomic instability. Most knowledge on the effect of ATM loss on DDR and tumor therapeutic response is from ataxia-telangiectasia (A-T) patients and ATM knockout models. A distinct and more deleterious phenotype has been proposed for kinase-dead (KD) ATM mutants that retain ATM protein expression. Inhibition of ATM activity does not fully mimic ATM loss; therefore, KD ATM mutants may be better predictors of response to ATM inhibitors and chronic effects of ATM inhibition. Using an inducible lentiviral CRISPR/Cas9 genome-editing system, we generated in-frame ATM mutated clonal lines derived from a bladder cancer PDX model (BLX1), via targeting of ATM exon 57, which maps to the second exon in the kinase domain. CRISPR-edited clones consistently exhibited reduced phosphorylation of Kap1 (S824) but not CHK2, p53 or NBS1 when challenged with DSB-inducing agents such as ionizing radiation (IR) and etoposide. Similar results were obtained in unedited cells treated with the ATM inhibitor KU55933, confirming that changes observed are ATM-dependent and suggesting that reduced Kap1 phosphorylation is a robust readout of compromised ATM activity in response to DSB-inducing therapies. Radiosensitivity is a hallmark of the A-T syndrome and the CRISPR-edited clones had increased sensitivity to DSB-inducing agents including IR, bleomycin, etoposide, and doxorubicin but not to S-phase drugs gemcitabine and camptothecin in a 10-day colony formation assay. ATM deficiency increased the dependency of cancer cells on ATM-independent repair mechanisms. Indeed, short-term (4-day) proliferation assays demonstrated the synthetic lethality of KD ATM with ATR inhibitors (AZD6738, VE821), Chk1 inhibitors (MK8776, LY2606368), as well as high concentrations of PARP inhibitors (Olaparib, BMN673) but not the DNAPK inhibitor (NU7441). KD ATM clones did not have increased response to cisplatin as a single agent except at high concentrations (>10 µM); the combination of low-concentration cisplatin (<1 µM) and an ATR inhibitor had a greater effect in KD ATM clones compared to unedited cells, demonstrating increased dependence on ATR. In summary, CRISPR-Cas9 editing of the ATM kinase domain was a useful tool for characterizing ATM loss-of-function effects, identifying targets to resensitize ATM mutants and elucidating compensatory mechanisms driven by chronic ATM loss. Funded by NCI Contract No. HHSN261200800001E. This research was supported, in part, by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Citation Format: Lara H. El Touny, John Connelly, Curtis Hose, Anne Monks, Dianne Newton, Luke Stockwin, Melinda Hollingshead, Ralph Parchment, James H. Doroshow, Beverly A. Teicher, Annamaria Rapisarda. Modeling ATM loss of function via CRISPR-Cas9 as a predictive tool for therapeutic responses in cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A117.

Dr. Peter Ujhazy: research for the greater good

Peter Ujhazy ( Figure 1 ) is the Deputy Associate Director of the Translational Research Program, Division of Cancer Treatment and Diagnosis at the National Cancer Institute.

411TiP - First-in-human trial of 4'-thio-2'-deoxycytidine (TdCyd) in patients with advanced solid tumors

Methylation-mediated silencing of genes is an epigenetic mechanism implicated in tumorigenesis. Hypermethylation of cytosines at CpG sites in the regulatory sequences of tumor suppressor genes silences them in a manner akin to inactivating mutations. Agents that inhibit methylation are of clinical interest because of their potential to re-activate silenced tumor suppressor genes, and two DNA hypomethylating nucleosides have been approved by the FDA for the treatment of patients (pts) with hematologic malignancies. The nucleoside analog 4'-thio-2'-deoxycytidine (TdCyd, NSC764276) is a cytidine analog in which the 4'-O moiety in deoxyribose has been replaced with sulfur where it engages the active site of DNA methyltransferase I (DNMT1), a maintenance methyltransferase that contributes to the hypermethylation and silencing of tumor suppressor genes. TdCyd and its active metabolites are phosphorylated and incorporated into the DNA of human cells in culture and in xenografts at rates exceeding those of the natural nucleoside or ara-analogs. Growth inhibition and tumor shrinkage were found during TdCyd dosing in several human tumour xenografts. Depletion of DNMT1 was observed also in certain xenograft models. Trial design: We are conducting an open label phase 1 first-in-human trial of TdCyd, following an accelerated titration design. TdCyd is administered PO qd x 5 days of each week for 2 weeks in 21-day cycles; the primary objective of the study is to establish its safety, tolerability, maximum tolerated dose, and recommended phase 2 dose. The secondary objectives of the study of oral TdCyd include preliminary efficacy assessment, pharmacokinetic (PK) characterization and exploratory pharmacodynamic (PD) evaluation of re-expression of select genes silenced by methylation in circulating tumor cells (CTCs). Eligibility criteria: solid tumor pts whose disease has progressed on standard therapy, ECOG ≤ 2, and normal organ function. Blood samples for PK/PD analyses and gene expression in CTCs are being evaluated. Currently, we are enrolling at DL5 in the escalation portion of the trial. Clinical trial identification: NCT02423057 Legal entity responsible for the study: Division of Cancer Treatment and Diagnosis, National Cancer Institute

Abstract 1350: New NCI experimental therapeutics program (NExT) small molecule libraries for academic investigator hts projects

Abstract The NCI Experimental Therapeutics Program (NExT) is making available two new smaller (3.5K) screening libraries suitable for academic investigator HTS drug discovery programs. The NExT program is a partnership between NCI's Division of Cancer Treatment and Diagnosis (DCTD) and the Center for Cancer Research (CCR). NExT consolidates NCI's anticancer drug discovery and development resources with the goal of maintaining a robust and balanced therapeutics pipeline. It encompasses tasks from new target validation through Phase III clinical trial evaluation. The Program is designed to streamline development and testing of promising new anticancer drugs and to expedite their delivery to the bedside. As part of the Program to support HTS drug discovery, NCI has previously offered academic oncology investigators access to our full 83K NExT Diversity Library in single-use, 384-well plate format. The set was designed to identify lead small molecules for drug discovery programs. We are now making available 2 smaller pre-plated subsets of this library, the NExT Diversity 3500 and NExT Diversity 3500 SAR, for when screening the full set is not appropriate. Each library contains 3,500 compounds: NExT Diversity 3500 is a diverse sampling across the entire 83K library while the NExT Diversity 3500 SAR is designed to facilitate rapid SAR follow-up by sampling only those compounds that have at least 5 close neighbors in the 83K library. The new libraries were designed by initially filtering the full NExT Diversity Library for undesirable compounds such as PAINS. Both atom-pair based 2D pharmacophore fingerprints and ECFP6 topological fingerprints from ChemAxon were then employed to generate the two non-overlapping subset libraries. The NExT screening libraries are provided as a resource to academic oncology investigators through the NExT Program (see NExT Resources: http://next.cancer.gov/). The design details and physiochemical characteristics of the two new sets, NExT Diversity 3500 and NExT 3500 SAR, and procedures and criteria for accessing this resource are presented along with several other screening sets available through the Developmental Therapeutics Program (DTP). Citation Format: Raj N. Misra, Michael Eckert, Christian Laggner. New NCI experimental therapeutics program (NExT) small molecule libraries for academic investigator hts projects. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1350.

Abstract 5178: Determinants of response to temozolomide in an exceptionally sensitive patient derived model

Abstract Application of precision medicine to cancer treatment utilizes cutting-edge genomic sequencing techniques to identify specific mutations in tumors that can be matched to targeted therapies designed to treat those abnormalities. To complement NCI-MPACT, an ongoing molecular profiling-based clinical trial (NCT01827384), we used cell lines developed from several patient-derived xenograft (PDX) models to examine response to, and potential biomarkers for, the regimens of the 4-arm MPACT trial: veliparib (VLP)/temozolomide (TMZ), AZD1775/carboplatin, everolimus and trametinib. In vitro sensitivity of the PDX-derived cell lines to clinically achievable concentrations of these MPACT drugs (combinations and single agents) was examined in classic 2D cultures (monolayer on plastic) and 3D cultures (spheroids generated in ultra-low attachment culture plates). Responses in 2D and 3D cultures were similar after 7 days of drug exposure. Moreover, adding VLP (1.7μM or 5μM) in combination with TMZ did not enhance TMZ cytotoxicity. A bladder cancer cell line developed from a PDX model (BLX) showed exceptional sensitivity to TMZ (IC50 ∼ 3-5μM) compared to a lung cancer cell line (also produced from a PDX; LUX) which was insensitive (IC50 >40 μM). Loss of MGMT expression in glioma and possibly in colorectal carcinoma is considered a predictive biomarker for response to alkylating agents, such as dacarbazine and TMZ. Indeed, we could not detect MGMT protein expression in BLX, while MGMT was present at high levels in LUX. To elucidate unique determinants of BLX hypersensitivity to TMZ beyond MGMT expression, we examined DNA damage responses elicited in this model. Under both 2D and 3D conditions, exposure to TMZ (13 and 40μM) for various times (4, 8, 12, 24, 48, 72 and 96 hrs) induced γH2AX after 24 hr, while PARP1 cleavage was induced as early as 48 hrs after drug addition indicating the onset of apoptosis. TMZ activated ATR and ATM signaling pathways especially at the later time points, paralleling the pharmacodynamics of PARP cleavage. Moreover, concordant with the sensitivity profiles, signaling activation in 2D and 3D conditions was similar. In contrast, none of these pathways were activated in the TMZ non-responsive LUX model. Cell line models developed from PDXs with intermediate MGMT expression are being evaluated. A key to success of personalized medicine in oncology will be the identification of genomic determinants that predict which individual cases will show exceptional responses to particular treatments, and our data suggest that PDX cell line models may be valuable for elucidating molecular and genetic characteristics of response to specific drugs and for the identification of predictive biomarkers. Funded by NCI Contract No. HHSN261200800001E. This research was supported, in part, by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Citation Format: Lara H. el Touny, John Connelly, Curtis Hose, Anne Monks, Mark W. Burkett, Erik Harris, Rene’ M. Delosh, Julie Laudeman, Chad Ogle, Russell Reinhart, Michael Selby, Thomas Silvers, David Evans, Dianne Newton, Luke Stockwin, Melinda Hollingshead, Ralph Parchment, James H. Doroshow, Beverly Teicher, Annamaria Rapisarda. Determinants of response to temozolomide in an exceptionally sensitive patient derived model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5178.

The death of cancer: after fifty years on the front lines of medicine, a pioneering oncologist reveals why the war on cancer is winnable-and how we can get there (devita, v. and devita-raeburn, e.; 2015) [Book Review

In this memoir of front-line action in the "war on cancer," the authors combine a survey of cancer biology with a recipe for more effective and personalized treatment of the myriad forms of cancer. Presented in chronological order in a smoothly written volume, the book tracks Dr. DeVita's early work as a clinical associate in the Division of Cancer Treatment (DCT) at the National Cancer Institute (NCI) through his pioneering treatment of Hodgkin's disease, and ultimately to his leadership of the NCI, and later of the Cancer Centers at Memorial Sloan Kettering in New York and at Yale in New Haven, Connecticut. Although at times a polemic against the Food and Drug Administration (FDA) for restricting patient access to new drugs, the book provides a vigorous defense for the "success" of hard-won advances in cancer treatment, such as improved cure rates and reduced mortality across the spectrum of all cancers, despite the huge allocation of effort and money (US$30 billion). In fact, it is the "boots on the ground" perspective of just how hard-won these advances have been that distinguishes this book from the other excellent book on this topic (The Emperor of All Maladies, by Siddhartha Mukherjee). Together, the two books provide a balanced perspective on the challenges that both patient and physician face when confronting cancer.

Abstract 5279: Establishing robust pharmacodynamic (PD) immunofluorescence assays of clinical biopsies at the National Cancer Institute: Optimized quality control procedures for the evaluation of DNA damage response and epithelial-mesenchymal transition (EMT) biomarkers

Abstract Robust PD assay results are valuable for informing decisions about continued preclinical and clinical development of new agents and for identifying effective combinations of targeted agents. The National Cancer Institute's Division of Cancer Treatment and Diagnosis (DCTD) develops and validates PD assays to obtain accurate information about drug effect on intended molecular targets in first-in-human clinical trials. Our group utilizes quantitative immunofluorescence assays (qIFA) of PD biomarkers in FFPE slides prepared from pre- and post-dose tumor biopsies collected from patients on early phase clinical trials. Paired biopsies are fixed and paraffin embedded, in parallel with tissue controls, such that all tissues are sectioned onto the same slide. Stringent methods to maintain the biopsy orientation are utilized during fixation, blocking and microtomy. A large number of sections are generated to increase the likelihood of finding optimal tumor regions of interest for biomarker analyses. Flanking H&E slides are utilized to assess the quality of the tumor biopsies. Whole slide scans of the H&E slides are shared with a clinical pathologist who determines whether the sections are sufficient or insufficient for the intended analysis. The pathology review allows the assay operator to select the optimal range of slides to stain and quantitatively analyze for the biomarker(s) of interest. Pathology-guided regions allow the operator to focus on tumor regions of interest and avoid normal tissue and/or confounding regions compromised by handling and processing artifacts. For nuclear biomarkers such as γH2AX and related DNA damage response biomarkers the pathologist annotates areas of sufficient tumor content and viability. For biomarker changes in tissue architecture such as that seen in Epithelial Mesenchymal Transition biomarkers, including E-Cadherin, Vimentin and β-catenin, the pathology annotation denotes areas of normal tissue and necrosis to eliminate from the qIFA analysis. All other areas of sufficient tumor cellularity from the entire slide are evaluated for the EMT biomarkers. Use of the pathology-annotated whole slide image as a tool for guidance of the operator performing the quantitative evaluation of the PD biomarker helps to ensure a non-subjective analysis. Finally, steps are taken to ensure proper storage of the paraffin slides, including paraffin dipping after microtomy, which is critical for preservation of labile epitopes such as phosphorylated proteins. We will present details of these optimized methods as well as key lessons learned during the preclinical and clinical implementation of qIFA measurements of PD biomarkers for various molecular targeted agents. Funded by NCI Contract No HHSN261200800001E. Citation Format: Katherine V. Ferry-Galow, Scott M. Lawrence, Tony Navas, Hala R. Makhlouf, Donna O. Butcher, Brad A. Gouker, William H. Yutzy, Jiuping Ji, Robert Kinders, Ralph E. Parchment, Shivaani Kummar, Joseph E. Tomaszewski, James H. Doroshow. Establishing robust pharmacodynamic (PD) immunofluorescence assays of clinical biopsies at the National Cancer Institute: Optimized quality control procedures for the evaluation of DNA damage response and epithelial-mesenchymal transition (EMT) biomarkers [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5279. doi:10.1158/1538-7445.AM2015-5279

Abstract 5139: Cell sorting strategies for the isolation of cancer cells and associated fibroblasts from tumor biopsies, surgical resections, and patient-derived xenografts

Abstract The ability of early passage patient-derived tumor xenografts (PDXs) to recapitulate human tumor biology makes them invaluable tools for pre-clinical drug development (1, 2). Similarly, clinically-annotated human tumor cell lines of low passage have potential as scalable biosimilar reagents for in vitro study. Here, we describe an integrated platform for the purification, expansion, and quality control of tumor cells and cancer-associated fibroblasts (CAFs) from patient biopsies, surgical resections, and mouse PDX models. Following dissociation of solid tumors, cells are cultured on Matrigel® in fibroblast conditioned media containing Y-27632 (a Rho-associated kinase inhibitor). Cells are then analyzed by FACS using a panel of species- or cell type-specific antibodies. This panel (anti-mCD9, hCD9, hCD24, hCD90, MHC, HLA, and hEPCAM) permits quantitation of the percentage of mouse/human cells, and the percentages of tumor cells and CAFs in each patient specimen. From these data, a cell sorting strategy can be selected (e.g. mCD9 and hCD90 with hEpCAM or CD24) that will permit purification of both tumor cells and CAFs, along with removal of any mouse fibroblast contamination (for PDX tumors). Samples are sorted multiple times until target populations reach 99.99% purity (typically 2-3 rounds). Purified cells are then returned to culture, expanded over several weeks, and frozen at low passage. The purity and identity of these cultures is quality-assured using 1) mouse-specific QPCR, 2) a cDNA array designed to differentiate fibroblasts from tumor cells, and 3) STR genotyping. Tumor lines and CAFs are then scaled and re-qualified at passages 10 and 20, an essential step given the propensity of low-frequency mouse and human fibroblasts to ‘overtake’ impure cultures with time. Lastly, cancer lines and CAF cultures undergo genomic characterization and are evaluated for in vivo tumorigenicity (or the lack thereof for CAFs). To date, the platform has been used to generate a growing number of purified cancer and CAF pairs from patient materials. In conclusion, our goal is to provide useful reagents to the extramural community that will accompany current efforts within the Division of Cancer Treatment and Diagnosis (DCTD, NCI) aimed at generating a large and extensively characterized repository of PDX models. Funded by NCI Contract No. HHSN261200800001E.

Funding opportunities in the NCI'S cancer diagnosis program

The Cancer Diagnosis Program is part of the NCI's Division of Cancer Treatment and Diagnosis and supports a variety of funding opportunities related to cancer diagnosis and treatment. New funding opportunities include: NCI's IMAT Program, Academic-Industrial Partnerships for Translation of Technologies for Cancer Diagnosis and Treatment, Assay Validation for High Quality Markers for NCI-Supported Clinical Trials, Opportunities in Bio-engineering Grants and Advancing Translational and Clinical Probiotic/Prebiotic and Human Microbiome Research. These and other related funding opportunities will be presented.

453 Plasma metabolomic signature of novel signal transduction inhibitors from preclinical identification to clinical validation

function aMOI and a NF1 loss of function aMOI, while SW694 has a NF1 loss of function aMOI, but these mutations do not correlate with sensitivity to trametinib. Interestingly, HT1080 has a heterozygous mutation in NRAS (Q61K) and mutations in NRAS have been shown to correlate with sensitivity to trametinib in cell lines with BRAF wild type background. To validate whether NRAS Q61K determines sensitivity to trametinib in HT1080, we used the CRISPR/Cas9 system to repair NRAS Q61K by homologous recombination. The system utilizes the type II prokaryotic CRISPR/Cas9 adaptive immune system and targets the Cas9 nuclease by a 20 nucleotides guide sequence cloned upstream of a 50-NGG “protospacer adjacent motif” (PAM). The induced site-specific strand breaks are repaired either by nonhomologous end-joining (NHEJ) to yield indels or by homologous recombination (HR) if homologous donor templates are available, thereby enhancing the efficiency of HR-based gene targeting. We designed several different small guide RNA sequences and we verified that all sequences were able to introduce indels in the NRAS gene using a control cell line. We therefore used either wild type Cas9 or Cas9D10A (nickase) with donor DNAs to perform NRAS gene repair. Clones are actively being screened for NRAS repair. Results for these experiments will be presented in the poster. The combination of drug sensitivity data, together with the mutation sequencing and precise gene editing using the CRISPR/Cas9 system may lead to precise targeting of genomic errors and to the identification of more effective therapeutic strategies for sarcoma. Funded by NCI Contract No. HHSN261200800001E. This research was supported [in part] by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute.

Abstract 3741: Development and characterization of a cell based assay for the validation of mutant IDH1 inhibitors

Abstract Recurrent isocitrate dehydrogenase (IDH) mutations have recently been identified in several cancer types. These point mutations specifically affect IDH1 and IDH2 active site arginine residues and confer the ability to reduce α-ketoglutarate to R-2-hydroxyglutarate (2HG), an oncometabolite that competitively inhibits α-ketoglutarate-dependent enzymes, such as histone and DNA demethylases. Accumulating evidence indicates that mIDH enzymes are attractive targets for the development of novel cancer therapeutics. To validate potential mIDH inhibitors in a cellular assay, we acquired several cell lines that harbor mIDH1 R132H and R132C: HCT116 mIDH1 (colon cancer, mIDH1 R132H +/-, Horizon Discovery), U87 MG mIDH1 (glioma, m IDH1 R132H +/+, Japan), HT1080 (fibrosarcoma, mIDH1 R132C +/-) and JJ012 (chondrosarcoma, mIDH1 R132C +/-, Rush University Medical Center). Cell lines were sequenced to confirm the presence of mIDH1 and 2HG production was measured in cellular supernatants by GC-MS. We observed that cells harboring mIDH1 R132C produce higher amounts of 2HG compared to lines harboring mIDH1 R132H, in agreement with recent reports. In addition, we show that accumulation of 2HG is time dependent and that its half-life in cellular supernatant is over 48 hours. Moreover, we demonstrate that 2HG is a stable analyte in media (through five sample free-thaw cycles) and that our samples are within linear range, providing a good intra-assay and inter-assay variability (CV <10%). Using this assay we evaluated the impact of five reference compounds obtained from published structures (Agios Pharmaceuticals, Cambridge MA), on the accumulation of 2HG in the supernatant of HT1080. These compounds were reported to have different ranges of activity against mIDH1 R132H and R132C enzymes in in vitro assays. Four of the mIDH reference compounds significantly inhibited 2HG accumulation in HT1080 supernatants in a dose dependent fashion, with potencies that were comparable to enzyme inhibition values. In agreement with previous reports, none of compounds affected HT1080 proliferation over a 48 hours incubation. In conclusion, we have identified mIDH cellular models that produce differing concentrations of 2HG, detectable by a GC/MS assay with sensitivity sufficient to determine levels as low as 0.1μg/ml. We have tested several inhibitors of the mIDH enzyme and confirmed an association between in vitro enzyme inhibition and a decrease in 2HG accumulation; therefore this assay can be used for the cellular evaluation of novel mIDH1 inhibitors. Funded by NCI Contract No. HHSN261200800001E. This research was supported [in part] by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Citation Format: Nicole D. Fer, Erik Harris, Stephen Fox, Ming Zhou, Catherine Simpson, Jing Liu, Ilia Korboukh, Emily A. Hull-Ryde, William P. Janzen, Stephen V. Frye, Anne Monks, Beverly Teicher, Annamaria Rapisarda. Development and characterization of a cell based assay for the validation of mutant IDH1 inhibitors. [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 3741. doi:10.1158/1538-7445.AM2014-3741