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Ras proto-oncogenes and numerous effectors of Ras signaling are recurrently mutated in acute myeloid leukemia (AML). While Ras represents an attractive therapeutic target, attempts at targeting oncogenic Ras directly have not been successful. To evaluate potential mechanisms of resistance to Ras targeted treatment in AML, our group leveraged a robust genetically-engineered mouse model in which AML is driven by expression of a tetracycline-repressible, constitutively active NRAS oncogene, tre-NRAS(V12), and a Mll-AF9 “knock-in” leukemogenic fusion gene (tre-NRAS(V12) & Mll-AF9 or tNM AML). The leukemia cells in this model are “addicted” to NRAS(V12), and mimicking Ras targeted treatment by doxycycline (Dox)-mediated suppression of the tre-NRAS(V12) oncogene expression results in rapid eradication of AML cells in vitro and in vivo. Furthermore, prolonged suppression of NRAS(V12) expression in NRAS(V12)-dependent (NRD) AML leads to the spontaneous development of NRAS(V12)-independent (NRI), “relapsed/refractory”, AML in about half of Dox treated mice. To elucidate the mechanism(s) that lead to the development of “relapsed/refractory” NRI AML following suppression of NRAS(V12) we performed transcriptome-wide next generation RNA-sequencing and comprehensive flow cytometric analysis of cancer signaling pathways comparing the parental NRD AML and two NRI AMLs. We confirmed that the NRAS(V12) oncogene is not aberrantly re-expressed. Furthermore, endogenous Ras gene expression is not up-regulated, and there is no evidence of reactivation of canonical Ras effector signaling pathways in either of the NRI AMLs by phospho-flow cytometry. Our preliminary analysis of cancer signaling pathways and transcriptome-wide RNA sequencing have identified candidate mediators of NRAS(V12)-independent AML growth and survival including the Myb proto-oncogene and anti-apoptotic Bcl2 that are enriched in both NRI AMLs relative to NRD AML at both the transcript and protein level. Furthermore, Myc protein is enriched in both NRI AMLs compared to NRD AML. We are currently performing a more comprehensive analysis of our next-generation RNA sequencing data to refine our list of candidate genes, and investigating the potential functional roles of Bcl2, Myb, and Myc in the development of NRI AML. In our initial functional studies, inhibition of Bcl2 activity suppresses NRI AML leukemic colony formation in vitro, and we are currently evaluating the ability of enforced expression of Bcl2, Myb, or Myc in NRD AML cells to render them NRAS(V12)-independent. To further investigate the translational potential of our findings, we are also evaluating the anti-leukemic effect of clinically relevant inhibitors of Bcl2 with inhibitors of canonical Ras effector pathways (RAF-MEK-ERK and PI3K-AKT-mTOR) in preclinical AML models. In this way we hope to gain a better understanding of mechanisms of treatment resistance to Ras targeted therapies, and thereby provide a foundation for the rational development of novel targeted treatment approaches for AML. Citation Format: Craig E. Eckfeldt, Robin DW Lee, Emily J. Pomeroy, Alpay N. Temiz, Susan K. Rathe, Jing Ma, Tanja A. Gruber, Ernesto Diaz-Flores, James R. Downing, Kevin M. Shannon, David A. Largaespada. Mechanisms of treatment resistance following Ras targeted therapy in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B01.

DNA sequence motifs with the ability to form non-B (non-canonical) structures have been linked to a variety of regulatory and pathological processes. Although the exact mechanism is unknown, recent work has provided significant evidence that non-B DNA structures may play a role in DNA instability and mutagenesis, leading to both DNA rearrangements and increased mutational rates, which are hallmarks of cancer. We have developed algorithms to identify a wide variety of non-B-DNA-forming motifs, including G-quadruplex-forming repeats, direct repeats and slipped motifs, inverted repeats and cruciform motifs, mirror repeats and triplex motifs, and A-phased repeats. After identifying these motifs in the mammalian reference genomes of human, mouse, chimpanzee, macaque, cow, dog, rat and platypus, the data were made publicly available in non-B DB [1]. However, it soon became apparent that it was not feasible to annotate the ever-growing list of genomic data and that it would be more effective to provide researchers with a systematic tool to predict these motifs in their own genomic data. Thus, the non-B DNA Motif Search Tool (nBMST) was created, and it is freely available online [2]. nBMST is a web interface that enables researchers to interactively submit any DNA sequence for searching for non-B DNA motifs. Once a user submits one or more DNA sequences in FASTA format, nBMST returns a comprehensive results page that contains the following: downloadable files in both a tab-delimited format and a generic feature format (GFF); a visualization, including PNG images; and a dynamic genome browser created using the Generic Genome Browser (GBrowse) [3] (version 2.0). Currently, nBMST allows file sizes of up to 20 MB of DNA sequence to be uploaded and stores the results for registered users for up to six months. In summary, the purpose of nBMST is to help provide insight into the involvement of alternative DNA conformations in cancer and other diseases, as well as into other potential biological functions.