Research
Targeting the Palmitoylation/Depalmitoylation Cycle in NRAS Mutant Cancers
Somatic NRAS mutations are highly prevalent in acute myeloid leukemia (AML), melanoma and other cancers. While challenging, directly inhibiting the biochemical output of oncogenic Ras proteins is the most rational strategy for treating RAS mutant cancers. We are working to therapeutically target N-Ras depalmitoylation and repalmitoylation in hematologic malignancies and other cancers. Because the normal K-RAS4b isoform does not require palmitate turnover, this approach has the potential to be selective for cancer cells that are dependent on oncogenic NRAS. In collaboration with Ben Cravatt (Scripps Research Institute) and Micah Niphakis (Lundbeck), we are characterizing how palmitoylation/depalmitoylation/repalmitoylation regulates normal and oncogenic N-Ras function and are testing chemical inhibitors of enzymes responsible for N-Ras depalmitoylation. We are complementing these cell and chemical biology studies with genetic experiments to verify the relevant biological targets of small-molecule activity, as well as the general viability of these enzymes as therapeutic targets for further drug discovery.
Selected Publications
Remsberg JR, Suciu RM, Zambetti NM, Hanigan TW, Firestone A, Inguva A, Long A, Ngo N, Lum KM, Henry CL, Predovic M, Huang B, Howell AR, Niphakis MJ, Shannon K, and Cravatt BF. ABHD17 enzymes regulate dynamic plasma membrane palmitoylation and N-Ras-dependent cancer growth. Nat Chem Biol (in revision) and preprint: https://biorxiv.org/cgi/content/short/2020.05.21.108316v1.
Zambetti NA, Firestone AJ, Remsberg JR, Huang BJ, Wong JC, Long AM, Predovic M, Suciu RM, Inguva A, Kogan SC, Haigis KM, Cravatt BF, Shannon K. Genetic disruption of N-RasG12D palmitoylation perturbs hematopoiesis and prevents myeloid transformation in mice. Blood 2020; 135:1772-178.
Xu J, Hedberg C, Dekker FJ, Li Q, Haigis KM, Hwang E, Waldmann H, Shannon K. Inhibiting the palmitoylation/depalmitoylation cycle selectively reduces the growth of hematopoietic cells expressing oncogenic Nras. Blood 2012; 119:1032-5.
Mechanisms of Response and Resistance in AMLs Characterized by Hyperactive Ras Signaling
Somatic NRAS and KRAS mutations or NF1 inactivation occur in 20-25% of acute myeloid leukemias (AMLs) and are particularly common in the pediatric age group. We performed retroviral insertional mutagenesis in Nf1, Nras, and Kras mutant mice to generate genetically diverse panels of transplantable, primary AMLs characterized by hyperactive Ras signaling. We then treated cohorts of recipient mice transplanted with independent primary AMLs in vivo with kinase inhibitors targeting downstream Ras effector pathways alone and in combination with chemotherapy or other targeted therapies. A subset of these AMLs responded dramatically to different treatments in vivo, but ultimately acquired drug resistance and relapsed. We are continuing to characterize mechanisms of drug resistance, identifying pathways that modulate sensitivity to individual agents and drug combinations, and extending this work to human patient derived xenograft (PDX) models of AML.
Selected Publications
Burgess MR, Hwang E, Mroue R, Bielski CM, Wandler AM, Huang BJ, Firestone AJ, Young A, Lacap JA, Crocker L, Asthana S, Davis EM, Xu J, Akagi K, Le Beau MM, Li Q, Haley B, Stokoe D, Sampath D, Taylor BS, Evangelista M, Shannon K. KRAS allelic imbalance enhances fitness and modulates MAP kinase dependence in cancer. Cell 2017; 168:817-29 e15.
Burgess MR, Hwang E, Firestone AJ, Huang T, Xu J, Zuber J, Bohin N, Wen T, Kogan SC, Haigis KM, Sampath D, Lowe S, Shannon K, Li Q. Preclinical efficacy of MEK inhibition in Nras-mutant AML. Blood 2014; 124:3947-55.
Ward AF, Braun BS, Shannon KM. Targeting oncogenic Ras signaling in hematologic malignancies. Blood 2012; 120:3397-406.
Li Q, Haigis KM, McDaniel A, Harding-Theobald E, Kogan SC, Akagi K, Wong JC, Braun BS, Wolff L, Jacks T, Shannon K. Hematopoiesis and leukemogenesis in mice expressing oncogenic NrasG12D from the endogenous locus. Blood 2011; 117:2022-32.
Lauchle JO, Kim D, Le DT, Akagi K, Crone M, Krisman K, Warner K, Bonifas JM, Li Q, Coakley KM, Diaz-Flores E, Gorman M, Przybranowski S, Tran M, Kogan SC, Roose JP, Copeland NG, Jenkins NA, Parada L, Wolff L, Sebolt-Leopold J, Shannon K. Response and resistance to MEK inhibition in leukaemias initiated by hyperactive Ras. Nature 2009; 461(7262):411-4.
Intrinsic and Acquired Drug Resistance in T-cell Acute Lymphoblastic Leukemia
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer characterized by aberrant proliferation of T-lymphoblasts. Recent data revealed a high incidence of both Ras pathway and NOTCH1 mutations in T-ALLs, particularly early T precursor (ETP) ALL. Previous work in our lab using retroviral insertional mutagenesis (RIM) generated panels of transplantable T-ALLs in wild type and Kras mutant mice. Treating these mice with targeted inhibitors of Ras effector molecules uncovered variability with respect to initial responses and acquired resistance to therapy. We have recently extended this work to fully characterize the genetic alterations in these leukemias and to investigate mechanisms of intrinsic and acquired resistance to glucocorticoids, which are the most effective know treatment for ALL.
Selected Publications
Wandler AM, Huang BJ, Craig J, Hayes K, Yan H, Meyer LK, Scacchetti A, Monsalve G, Dail M, Li Q, Wong JC, Weinberg O, Hasserjian RP, Kogan SC, Jonsson P, Yamamoto K, Sampath D, Nakitandwe J, Downing JR, Zhang J, Aster J, Taylor BS, Shannon K. Loss of glucocorticoid receptor expression mediates dexamethasone resistance in T-cell acute lymphoblastic leukemia. Leukemia 2020; 34: 2025-2037.
Meyer LK, Huang BJ, Delgado-Martin C, Roy RP, Hechmer A, Wandler AM, Vincent TL, Fortina P, Olshen AB, Wood BL, Horton TM, Shannon KM, Teachey DT, Hermiston ML. Glucocorticoids paradoxically facilitate steroid resistance in T-cell acute lymphoblastic leukemias and thymocytes. J Clin Invest 2019; https://doi.org/10.1172/JCI130189.
Huang BJ, Wandler AM, Meyer LK, Dail M, Daemen A, Sampath D, Li Q, Wang X, Wong JC, Nakitandwe J, Downing JR, Zhang J, Taylor BS, Shannon K. Convergent genetic aberrations in murine and human T lineage acute lymphoblastic leukemias. PLoS Genet 2019;15(6):e1008168.
Dail M, Wong J, Lawrence J, O'Connor D, Nakitandwe J, Chen SC, Xu J, Lee LB, Akagi K, Li Q, Aster JC, Pear WS, Downing JR, Sampath D, Shannon K. Loss of oncogenic Notch1 with resistance to a PI3K inhibitor in T-cell leukaemia. Nature 2014;513(7519):512-6.
Dail M, Li Q, McDaniel A, Wong J, Akagi K, Huang B, Kang HC, Kogan SC, Shokat K, Wolff L, Braun BS, Shannon K. Mutant Ikzf1, KrasG12D, and Notch1 cooperate in T lineage leukemogenesis and modulate responses to targeted agents. Proc Natl Acad Sci USA 2010;107:5106-11.
Novel Germline and Somatic KRAS Mutations in Developmental Disorders and Cancer
Codons 12, 13, and 61 of KRAS are the most common targets of oncogenic mutations in cancer. In 2006, we discovered germline KRAS mutations in children with Noonan syndrome and other Rasopathy disorders and showed that these alleles encode gain-of-function proteins that are less activated than oncogenic K-Ras. We have modeled two of these mutations (P34R and T58I) in mice, which provide robust platforms for performing biologic and preclinical studies. We have also characterized unusual somatic KRAS mutations identified in patients with hematologic diseases and generated mutant oncoproteins with “second site” amino acid substitutions to investigate the role of individual Ras effector pathways in development and tumorigenesis.
Selected Publications
Chen P-Y, Huang BJ, Harris M, Boone C, Wang W, Carias H, Mesiona B, Mavrici D, Kohler A, Bollag G, Zhang C, Zhang Y, Shannon K. Structural and functional analyses of a germline KRAS T50I mutation provide insights into Raf activation. JCI Insight. 2023 Sep 8;8(17):e168445. doi: 10.1172/jci.insight.168445.
Wong JC, Perez-Mancera PA, Huang TQ, Kim J, Grego-Bessa J, del pilar Alzamora M, Kogan SC, Sharir A, Keefe SH, Morales C, Schanze D, Zenker M, Sheppard D, Klein OD, Tuveson DA, Braun BS*, Shannon K*. KrasP34R and KrasT58I mutations induce distinct RASopathy phenotypes in mice. JCI Insight. 2020 Nov 5;5(21):e140495. doi: 10.1172/jci.insight.140495.
White Y, Bagchi A, Van Ziffle J, Inguva A, Bollag G, Zhang C, Carias H, Dickens D, Loh M, Shannon K, Firestone AJ. KRAS insertion mutations are oncogenic and exhibit distinct functional properties. Nat Commun 2016; 7:10647.
Shieh A, Ward AF, Donlan KL, Harding-Theobald ER, Xu J, Mullighan CG, Zhang C, Chen SC, Su X, Downing JR, Bollag GE, Shannon KM. Defective K-Ras oncoproteins overcome impaired effector activation to initiate leukemia in vivo. Blood 2013; 121:4884-93.
Schubbert S, Shannon K, Bollag G. Hyperactive Ras in developmental disorders and cancer. Nat Rev Cancer 2007; 7:295-308.
Schubbert S, Bollag G, Lyubynska N, Nguyen H, Kratz CP, Zenker M, Niemeyer CM, Molven A, Shannon K. Biochemical and functional characterization of germ line KRAS mutations. Mol Cell Biol 2007; 27:7765-70.
Schubbert S, Zenker M, Rowe SL, Boll S, Klein C, Bollag G, van der Burgt I, Musante L, Kalscheuer V, Wehner LE, Nguyen H, West B, Zhang KY, Sistermans E, Rauch A, Niemeyer CM, Shannon K, Kratz CP. Germline KRAS mutations cause Noonan syndrome. Nat Genet 2006; 38:331-6.
Functional Analysis of Leukemia-Associated Chromosome 7 Deletions and SAMD9/9L Mutations
Monosomy 7 (-7) and deletion 7q (del(7q)) are among the most common cytogenetic alterations found in myelodysplastic syndromes (MDS) and AML. Cytogenetic analysis of patients who developed myeloid malignancies with del(7q) uncovered a commonly deleted segment within chromosome band 7q22, suggesting that this region plays an important role in leukemogenesis. Genome sequencing of primary patient specimens showed that biallelic inactivation of individual 7q genes is remarkably infrequent in cases of AML or MDS with -7/del(7q). These data strongly support haploinsufficiency for multiple 7q genes as cooperating with mutations occurring elsewhere in the genome in the pathogenesis of MDS/AML. Myelodysplasia and Leukemia Syndrome with Monosomy 7 (MLSM7; OMIM 252270) is a familial cancer syndrome in which two of more siblings develop MDS or AML with loss of chromosome 7 in bone marrow cells. Surprisingly, MLSM7 does not follow the expected Knudson tumor suppressor model but was recently shown to result from germline gain-of-function SAMD9 and SAMD9L mutations. These functionally-related genes are located on chromosome band 7q11 and the mutant alleles are lost in the monosomy 7 bone marrows of affected patients due to a novel “adaptation by aneuploidy” mechanism. We used chromosome engineering to generate strains of mice with MB scale deletions in the DNA region syntenic to human chromosome 7q22 to model the consequences of chromosome 7 deletions in an in vivo setting and to develop accurate model systems for biologic and preclinical studies. We are characterizing these mice to understand the consequences of the loss of this region on hematopoiesis and leukemogenesis. A question of specific interest is how chromosome 7q22 deletions cooperate with germline inactivation of Samd9l (mice lack a Samd9 gene).
Selected Publications
Wong JC, Bryant V, Lamprecht T, Ma J, Walsh M, Schwartz J, Del Pilar Alzamora M, Mullighan CG, Loh ML, Ribeiro R, Downing JR, Carroll WL, Davis J, Gold S, Rogers PC, Israels S, Yanofsky R, Shannon K, Klco JM. Germline SAMD9 and SAMD9L mutations are associated with extensive genetic evolution and diverse hematologic outcomes. JCI Insight 2018;3(14), PMC6124395.
Wong JC, Weinfurtner KM, Alzamora Mdel P, Kogan SC, Burgess MR, Zhang Y, Nakitandwe J, Ma J, Cheng J, Chen SC, Ho TT, Flach J, Reynaud D, Passegue E, Downing JR, Shannon K. Functional evidence implicating chromosome 7q22 haploinsufficiency in myelodysplastic syndrome pathogenesis. Elife 2015;4. PMC4569895.
Wong JC, Zhang Y, Lieuw KH, Tran MT, Forgo E, Weinfurtner K, Alzamora P, Kogan SC, Akagi K, Wolff L, Le Beau MM, Killeen N, Shannon K. Use of chromosome engineering to model a segmental deletion of chromosome band 7q22 found in myeloid malignancies. Blood 2010; 115:4524-32.
Developmental and Hyperactive Ras SPORE
Drs. Shannon and D. Wade Clapp (Indiana University) hold a multiple Principal Investigator SPORE grant from the National Cancer Institute, which has the goal of conducting rational clinical trials in tumors characterized by NF1 gene mutations that are informed by basic research and preclinical studies in genetically accurate mouse models. With support from this SPORE grant, Dr. Shannon and his UCSF colleagues Ben Braun, Mignon Loh, and Elliot Stieglitz are investigating the efficacy of MEK inhibition and performing correlative molecular studies in children with relapsed/refractory juvenile myelomonocytic leukemia JMML. Please refer to the DHART SPORE website for additional information about this translational research program: https://www.dhartspore.org/.