Abstract: Background Deferasirox (DFX) is widely employed as iron chelation therapy (ICT) in the current clinical practice in patients with myelodysplastic syndromes (MDS) and chronic transfusion need. The efficacy of DFX in reducing median ferritin levels in different cohorts of these patients has been reported in many trials, but the lack of worldwide accepted criteria of individual response to ICT makes it difficult to appreciate its clinical relevance for any single patient. Aim To highlight the clinical impact of ICT with DFX in a large real-life cohort of MDS patients, based on different individual ferritin variation during treatment. Methods A retrospective cohort of 301 consecutive MDS patients [M/F 187/114 (62.1%/37.9%)] of any age followed in 20 hematological Centers in Italy was analyzed: the main features at diagnosis are reported in the Table 1. Individual response to ICT was categorized as complete response (CR) (ferritin levels 〈 500 ng/ml), partial response (PR) (ferritin levels 〈 1,000 ng/ml), ferritin improvement (FI) (ferritin reduction 〉 50% of baseline value but with levels 〉 1,000 ng/ml), ferritin stability (FS) (ferritin levels without changes from baseline during ICT) or no ferritin response (NR) (ferritin levels increasing during ICT). Results ICT was started after a median period from diagnosis and from transfusion start of 21.0 months [interquartile range (IQR) 8.9 - 44.3] and 11.3 months (IQR 7.1 - 21.7), respectively, with a median burden of red cell transfusions at baseline of 22 units (IQR 14 - 35). The main features of patients at baseline of ICT are reported in the Table 1. Starting DFX dose was 〈 10 mg/Kg in 38 patients (12.7%), 10 - 14 mg/Kg in 110 patients (36.6%), 15 - 19 mg/Kg in 57 patients (18.9%) and ≥ 20 mg/Kg in 96 patients (31.9%). As to individual response, 4 patients (1.3%) were too early for evaluation ( 〈 6 months of DFX treatment): in addition, 16 patients (5.4%) discontinued ICT behind 6 months from start, due to early toxicity (10 patients, 7 for gastro-intestinal toxicity and 3 for skin toxicity) or other reasons (unrelated death, AML evolution, transplant procedure). Among the remaining 281 patients, 37 (12.3%) achieved a CR, 65 (21.6%) a PR, 23 (7.6%) a FI, 112 (37.2%) a FS and 44 (14.6%) a NR. Five-year overall survival (OS) of the whole cohort from ICT start was 43.9% (95%CI 37.1 - 50.7). Five-year OS according to ICT response was 74.8% (95%CI 57.9 - 91.7) in patients with CR, 51.7% (95%CI 37.6 - 65.8) in patients with PR, 50.6% (95%CI 28.2 - 73.0) in patients with FI, 38.6% (95%CI 27.0 - 50.2) in patients with FS and 21.1% (95%CI 5.2 - 37.0) in patients with NR (p=0.002) (Figure 1). Five-year cumulative incidence of AML evolution (CIE) of the whole cohort from ICT start was 27.1% (95%CI 20.3 - 33.9). Five-year CIE according to ICT response was 7.6% (95%CI 0 - 18.0) in patients with CR, 27.0% (95%CI 13.0 - 40.5) in patients with PR, 38.3% (95%CI 15.5 - 61.7) in patients with FI, 20.8% (95%CI 10.4 - 31.2) in patients with FS and 57.7% (95%CI 31.9 - 83.5) in patients with NR (p=0.003) (Figure 2). Notably, no statistical difference was observed for both OS and CIE among patients achieving PR, FI or FS. Conclusions Present data highlight the clinical relevance of individual response in MDS patients receiving ICT with DFX. In particular, achievement of CR seemed related to a better OS and a lower CIE, while patients with NR had a significant worst OS and CIE: furthermore, the achievement of stable ferritin levels was associated with similar OS and CIE than PR and FI and thus should be considered as a response. Disclosures Latagliata: Celgene: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Oliva:Novartis: Consultancy, Speakers Bureau; Celgene Corporation: Consultancy, Honoraria, Speakers Bureau; Apellis: Consultancy. Pilo:Novartis: Other: Advisory board. Molteni:Celgene: Membership on an entity's Board of Directors or advisory committees. Balleari:Celgene: Membership on an entity's Board of Directors or advisory committees. Breccia:Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Incyte: Honoraria; Celgene: Honoraria. Foà:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Finelli:Novartis: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Celgene Corporation: Consultancy, Research Funding, Speakers Bureau.

Abstract: DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.

Abstract: CRISPR-based cancer dependency maps are accelerating advances in cancer precision medicine, but adequate functional maps are limited to the most common oncogenes. To identify opportunities for therapeutic intervention in other rarer subsets of cancer, we investigate the oncogene-specific dependencies conferred by the lung cancer oncogene, RIT1 . Here, genome-wide CRISPR screening in KRAS , EGFR , and RIT1 -mutant isogenic lung cancer cells identifies shared and unique vulnerabilities of each oncogene. Combining this genetic data with small-molecule sensitivity profiling, we identify a unique vulnerability of RIT1 -mutant cells to loss of spindle assembly checkpoint regulators. Oncogenic RIT1 M90I weakens the spindle assembly checkpoint and perturbs mitotic timing, resulting in sensitivity to Aurora A inhibition. In addition, we observe synergy between mutant RIT1 and activation of YAP1 in multiple models and frequent nuclear overexpression of YAP1 in human primary RIT1 -mutant lung tumors. These results provide a genome-wide atlas of oncogenic RIT1 functional interactions and identify components of the RAS pathway, spindle assembly checkpoint, and Hippo/YAP1 network as candidate therapeutic targets in RIT1 -mutant lung cancer.

Abstract: The role of PPM1D mutations in de novo gliomagenesis has not been systematically explored. Here we analyze whole genome sequences of 170 pediatric high-grade gliomas and find that truncating mutations in PPM1D that increase the stability of its phosphatase are clonal driver events in 11% of Diffuse Midline Gliomas (DMGs) and are enriched in primary pontine tumors. Through the development of DMG mouse models, we show that PPM1D mutations potentiate gliomagenesis and that PPM1D phosphatase activity is required for in vivo oncogenesis. Finally, we apply integrative phosphoproteomic and functional genomics assays and find that oncogenic effects of PPM1D truncation converge on regulators of cell cycle, DNA damage response, and p53 pathways, revealing therapeutic vulnerabilities including MDM2 inhibition.

Abstract: Chest pain and dyspnea are common symptoms in patients presenting to the emergency room (ER); oftentimes it is not possible to clearly identify the underlying cause, which may cause the patient to have to return to the ER. In other cases, while it is possible to identify the underlying cause, it is necessary to perform a large number of tests before being able to make a diagnosis. Over the last twenty years, emergency medicine physicians have had the possibility of using ultrasound to help them make and rule out diagnoses. Specific ultrasound tests have been designed to evaluate patients presenting with specific symptoms to ensure a fast, yet complete, evaluation. In this paper, we examine the role of ultrasound in helping physicians understand the etiology behind chest pain and dyspnea. We analyze the different diseases and disorders which may cause chest pain and dyspnea as symptoms and discuss the corresponding ultrasound findings.

Abstract: KRAS is the most commonly mutated oncogenes and is a major driver of tumor initiation and progression. Understanding the functional consequences of cancer-associated KRAS variants may have important clinical implications. For example, KRAS mutation status defines those that are likely to respond to EGFR-directed therapy in KRAS-mutant metastatic colorectal cancer. A compendium of all possible oncogenic KRAS alleles would serve as a roadmap for future therapeutic strategies directed at KRAS itself or downstream signaling effectors. Comprehensive mutagenesis of KRAS may also elucidate structure-function relationships that reveal novel biochemical properties that may be exploited for therapeutic gain. We performed saturation mutagenesis of both a wild-type (WT) and a G12D mutant form of KRAS cDNA and generated lentiviral expression libraries of 3,553 and 3,534 single amino acid substitution mutants of each backbone. We utilized these WT and G12D mutagenesis libraries for functional genetic screening to identify gain- and loss-of-function missense variants that alter critical oncogenic properties of KRAS. First, we sought to comprehensively identify all possible oncogenic missense mutations in KRAS that mediate oncogenic transformation. We stably transduced the WT library into immortalized human epithelial cells and evaluated growth in low attachment (GILA), an assay that is highly correlated with in vivo tumor formation. We identified all previously known hotspot oncogenic alleles of KRAS as well as many functionally relevant alleles that are also discovered at lower frequency in human tumors. Moreover, we also discovered a group of transforming KRAS variants that have not been well described in human tumors, thus revealing potentially novel activating mechanisms for oncogenic KRAS. In parallel, we utilized the G12D mutagenesis library to perform second-site suppressor screening to identify loss-of-function single amino acid changes that abrogate the transforming ability of oncogenic KRAS. We performed positive-selection screening in primary cell lines for variants that enable bypass of oncogene-induced senescence. Additionally, we conducted a negative-selection screen with the G12D library in a KRAS-dependent cancer cell line with inducible suppression of endogenous KRAS, thus identifying all possible second-site mutations that abolish KRAS-driven signaling necessary for maintenance of cellular proliferation and viability. Structure-function analysis of these data may reveal novel patterns of amino-acid changes that result in inactivation of oncogenic KRAS. In summary, this comprehensive dictionary of gain- and loss-of-function KRAS mutants will facilitate understanding of clinically important mutations and also yield novel insights into structure-function relationships that may improve our understanding of the KRAS oncogene. Citation Format: Eejung Kim, Seav Huong Ly, Nicole S. Persky, Belinda Wang, Xiaoping Yang, Federica Piccioni, Katherine Labella, Mihir Doshi, Robert E. Lintner, Cong Zhu, Scott Steelman, David E. Root, Cory M. Johannessen, Alex B. Burgin, Laura E. MacConaill, William C. Hahn, Andrew J. Aguirre. Saturation mutagenesis of KRAS reveals the functional landscape of missense variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 394. doi:10.1158/1538-7445.AM2017-394

Abstract: PDE3A-SLFN12 complex formation is induced by a class of compounds, now called “velcrins”, exemplified by the small molecule, DNMDP. Cancer cells that express elevated levels of PDE3A and SLFN12 are sensitive to a velcrin-mediated cytotoxic response, which is independent of PDE3A inhibition. However, the details of complex formation have not yet been revealed. We solved the crystal structure of PDE3A with a series of ligands bound to the active site and found that PDE3A exists as a dimer, and velcrin binding does not cause any obvious structural changes in the PDE3A protein structure. Hydrogen-deuterium exchange (HDX-MS) experiments with velcrin-bound PDE3A in the absence and presence of SLFN12 identified three regions of PDE3A that are shielded from solvent as a result of velcrin-induced SLFN12 binding. Two of these regions are near the velcrin binding site, and the third region lies at the PDE3A homodimerization interface. In order to further investigate the structural relationship between PDE3A, DNMDP, and SLFN12, we took a deep-mutation scanning (DMS) approach to identify residues of PDE3A that impact DNMDP sensitivity. A library of PDE3A alleles was developed in which the sequence encoding amino acids 668-1141, including the PDE3A catalytic domain, was substituted with a codon for every other possible amino acid or a stop codon in the context of the full-length cDNA. The library was transduced into PDE3A-knockout GB1 glioblastoma cells and assessed for survival in the presence of DMSO or DNMDP. Corroborating the HDX-MS data, we identified three regions of PDE3A in which missense mutations abrogated DNMDP response: the active site, the homodimerization surface, and an alpha helix containing amino acid F914. We confirmed that mutations of F914 and the homodimer interface retain the ability to bind resin-conjugated compound but fail to complex with SLFN12. Taken together, the HDX and DMS results suggest that PDE3A dimerization is required to stabilize velcrin-induced SLFN12 binding and implicate the alpha helix containing F914 as the SLFN12 binding interface of PDE3A. Citation Format: Xiaoyun Wu, Malvina Papanastasiou, Gavin Schnitzler, Colin Garvie, Stephanie Hoyt, Terry Zhang, James Mullahoo, Andrew Baker, Joseph McGaunn, Bethany Kaplan, Sooncheol Lee, Martin Lange, Steven Carr, Xiaoping Yang, Federica Piccioni, Andrew Cherniack, Matthew Meyerson, Heidi Greulich. Deep mutational scanning of PDE3A identifies residues required for DNMDP response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1219.