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1 Juvenile Myelomonocytic Leukemia: From Bedside to Bench and Back Mignon Loh, M.D. UCSF Benioff Professor of Children s Health Deborah and Arthur Ablin Chair of Pediatric Molecular Oncology Benioff Children s Hospital University of California, San Francisco PLEASE TURN OFF YOUR CELL PHONES Chair, Acute Lymphoblastic Leukemia Committee Children s Oncology Group There are two clinical challenges in JMML Disclosure of Relevant Financial Relationships 1. JMML is difficult to diagnose Category 1 Category 2 USCAP requires that all planners (Education Committee) in a position to influence or control the content of CME disclose any relevant financial relationship WITH COMMERCIAL INTERESTS which they or their spouse/partner have, or have had, within the past 12 months, which relates to the content of this educational activity and creates a conflict of interest. MIGNON LOH: NO RELEVANT FINANCIAL RELATIONSHIPS ALL of the following AMC >1/uL Blasts in PB/BM < 2% Absence of the t(9;22) or BCR/ABL fusion gene At least 2 of the following Circulating myeloid precursors WBC >1,/uL Increased Hgb F for age GM-CSF hypersensitivityudingon 7 Here s the second challenge 1. JMML is difficult to diagnose 2. JMML is hard to treat What strategies can we use to solve these problems? Genetics approach Identify novel genetic mutations Incorporate mutations into diagnostic criteria Correlate genotype with phenotype Category 1 Category 2 ALL of the following AMC >1/uL Blasts in PB/BM < 2% Absence of the t(9;22) or BCR/ABL fusion gene At least 2 of the following Circulating myeloid precursors WBC >1,/uL Increased Hgb F for age GM-CSF hypersensitivityudingon 7 1

2 What strategies can we use to solve these problems? Genetics approach Identify novel genetic mutations Incorporate mutations into diagnostic criteria Correlate genotype with phenotype Functional approach Determine how mutations contribute to pathogenesis Determine if perturbed signaling networks exist in JMML that can be harnessed therapeutically Use specific agents to reverse these abnormalities and test in preclinical models Take new agents into clinical trial The patients inform our science and we hope the science will lead to improved therapies for patients Juvenile myelomonocytic leukemia Juvenile myelomonocytic leukemia WHO classification: Mixed MDS/MPN of young children Shares clinical and biological features with CMML and CML Boys > girls Median age: 1.7 years Splenomegaly: 93% Lymphadenopathy: 59% WHO classification: Mixed MDS/MPN of young children Shares clinical and biological features with CMML and CML Boys > girls Median age: 1.7 years Splenomegaly: 93% Lymphadenopathy: 59% Survival following conventional chemotherapy is dismal Plt 33x1 9 /L, age 2 yrs:.8 Plt 33x1 9 /L, age 2 yrs:.5 Plt 33x1 9 /L, : Log Rank p=.1 years Niemeyer, Blood 1997 Juvenile myelomonocytic leukemia Juvenile myelomonocytic leukemia WHO classification: Mixed MDS/MPN of young children Shares clinical and biological features with CMML and CML Boys > girls Median age: 1.7 years Splenomegaly: 93% Lymphadenopathy: 59% Only hematopoietic stem cell transplantation is curative Relapse remains the most common cause of treatment failuretreatment failure Event free survival Years from study entry COG AAML122 WHO classification: Mixed MDS/MPN of young children Shares clinical and biological features with CMML and CML Boys > girls Median age: 1.7 years Splenomegaly: 93% Lymphadenopathy: 59% Hallmark laboratory feature is hypersensitivity of myeloid progenitors to CM-CSF Hematopoetic stem cell transplant is curative but relapse remains the most common cause of treatment failure Stieglitz, Peds. Blood and Cancer 215 Emanuel, Blood

3 Patients with NF1 are at Increased Risk of Developing Leukemia Leukemia Relative risk Confidence Interval CMML (JMML) ALL NHL Cooke, J. Pediatrics 1953 Neurofibromatosis Type 1 (NF1) NF1 Encodes a GTPase Activating Protein (GAP) dominant genetic disorder Spontaneous germline mutations can occur in up to 5% of cases pigmentary lesions benign and malignant tumors usually affect cells derived from the embryonic neural crest greatly increased risk of developing JMML rasgap (mammalian, p12) NF1 (mammalian, p28) IRA1/2 (S.cerevisiae, p32) GAP1 (Drosophila, p13) gap 1 (S.pombe, p8) Ras The beating heart of signal transduction Courtesy of F. McCormick, UCSF A. Wittinghofer 3

4 Ras Proteins Act as Molecular Switches NF1 associated JMML demonstrates NF1 is a classic tumor suppressor gene Mo Fa GL JMML Ward, Blood 212 Shannon, NEJM, 1994 Bollag, Nat. Genetics 1994 JMML is a disease of hyperactive Ras signaling Blood, April 1994 JAK STAT Grb2 SOS Shc Gab2 SHP 2 Ras GDP Ras GTP* Neurofibromin BCR ABL Effector Pathways Gene % NF1 15% RAS 25% Blood, November 1994 Noonan syndrome (Jackie Noonan, 1963) Pulmonic stenosis Missense somatic mutations in PTPN11 occur in de novo JMML Facial anomalies Myeloid disorders of Noonan Syndrome and a resident s rule recalled Shannon and Side, J. Pediatrics, 1997 Hypertrophic cardiomyopathy Nature Genetics 34, (23) Short stature Courtesy of Zenker/Kratz 4

5 JMML is a disease of hyperactive Ras signaling Harnessing SNP arrays demonstrated region on 11q with copy neutral LOH JAK STAT Grb2 SOS Shc Gab2 SHP 2 Ras GDP Ras GTP* Neurofibromin BCR ABL Gene % Effector Pathways NF1 15% RAS 25% PTPN11 (SHP2) 35% Loh, ML et al. Blood 29;114:1859 Analysis of deleted region suggested CBL as a likely candidate gene Wildtype Diagnosis Loh, ML et al. Blood 29;114: CBL mutations detected in 68 JMML patients without RAS/PTPN11/NF1 Location Nucleotide change Amino acid change No of Patients Intron G>C Splice site 1 Exon Tyr>His T>G 371 Tyr>Asp A>G 371 Tyr>Cys T>C 38 Leu>Pro T>C 381 Cys>Arg 1 115T>C 384 Cys>Arg T>C 396 Cys>Arg del99bp* deletion T>C 44 Cys>Arg T>C 48 Trp>Arg 1 Intron C>T* Splice site A>G Splice site 1 Exon G>T 415 Gly>Val 1 * 2 heterozygous mutations Loh, ML et al. Blood 29;114:1859 Is this a new germline syndrome? CBL Mutations in JMML are Frequently Inherited in an Autosomal Dominant Fashion Wildtype Lung Ca, 5s Diagnosis 6 mos. 6 mos. Cord Blood *7 mos. Loh, Blood, 29, 114: 1859 Niemeyer, Nature Genetics, 21, 42:

6 JMML is a disease of hyperactive Ras signaling Rasopathies Support Hypothesis that Pediatric Cancer is Development Gone Awry JAK STAT Grb2 SOS Shc Gab2 SHP 2 Ras GDP Ras GTP* Neurofibromin BCR ABL Gene % CBL Effector Pathways NF1 15% RAS 25% PTPN11 35% (SHP2) CBL 1% 214 by American Society of Hematology Chang, T Blood 214 Can Ras Signaling Networks be Therapeutically Harnessed? Can we harness perturbed signaling networks for novel therapeutics? JAK2 STAT5 Grb2 SOS Shc Gab2 SHP 2 CBL Ras GDP Ras GTP RAF Neurofibromin PI3K Ruxolitinib JAK2 STAT5 Grb2 SOS Shc Gab2 SHP 2 Ras GDP Ras GTP* RAF Neurofibromin PI3K Pictilisib Copanlisib Ral GDS Ral A MEK ERK Survival, Proliferation S6 Akt mtor PD32591 Trametinib Binimetinib Selumetinib Pimasertib Cobimetinib Ral GDS Ral A MEK ERK Survival, Proliferation S6 Akt mtor Dactolisib Sirolimus Everolimus Pre-clinical MPN mouse models PD32591 Results in MEK Inhibition In Vivo Mx1-Cre,Nf1 flox/flox (Parada, Shannon, Le) Mx1-Cre, Kras G12D (Jacks, Shannon, Braun) Mx1-Cre, Nras G12D (Jacks, Shannon, Haigis) Ptpn11 D61Y (Neel, Araki) Mx1 Cre, Nf1 flox/flox + PD mg/kg/day Nf1 flox/flox Nf1 wt Mx1-Cre, Ptpn11 E76K (Qu, Xu) ccbl C379A/- (Langdon, Thien, Rathinam) MMTV-Cre, Cbl flox/flox ;Cbl-b -/- (Band, Naramura) Myeloid progenitors No JMML cell lines, xenografts difficult to establish perk pstat5 Chang, T, JCI 213 6

7 PD32591 Reduces Myeloproliferation and Enhances Erythropoiesis in Mx1 Cre, Nf1 flox/flox Mice PD32591 Reduces Splenic Hematopoiesis In Vivo BFU-E CFU-E WBC (K/ L) % Reticulocytes 5 ** 4 3 ** *** Days From Enrollment 15 ** ** ** 1 5 HB (g/dl) Spleen weight (g) Days From Enrollment 5 *** Veh 91 Nf1 -/- Veh Nf1 -/- 91 Colony number e d e d Vehicle 91 Vehicle 91 Nf1 -/- Colony number W Nf1 -/- T Nf1 -/- Veh Nf1 -/- 91 d e d Vehicle 91 Vehicle 91 Veh 91 Nf1 -/- Veh Nf1 -/ Days From Enrollment Vehicle 91 Vehicle 91 Nf1 -/- Chang, JCI 213 Chang, JCI 213 Primary human JMML cells vs. MEK inhibition Trametinib % Maximal colony growth Maximal colony growth No drug (n=6) No drug (n=6) 1nM PD-91 (n=5) Normals Normals (n=13) (n=13) JMML JMML (n=23) (n=23) Selective allosteric inhibitor of MEK1/2 OG daily dose Long circulating t 1/2 Sustained suppression of perk1/2 for more than 24h FDA approved for melanoma MEK11654:Phase 1 in pediatric solid tumors currently enrolling GM-CSF GM-CSF dose dose J. Weng, Loh lab Borthakur, G, Cancer 216 ADVL1521 Will MEK inhibition Monotherapy Work? Part A: Phase 2 Trametinib In JMML Genotyping Population: Relapsed/Refractory JMML Age 2 years 21 years Colony Assays Endpoints: Objective Response Safety/Toxicity Pharmacodynamics PTPN11 PTPN11 Dose Level Trametinib 1 One dose < RP2D 1% RP2D What Additional Genetic or Epigenetic Modifiers Occur in JMML? 7

8 The grand fishing expedition? Genomic Landscape of JMML Stieglitz, Taylor Wiener, Nature Genetics 215 Stieglitz, Taylor Wiener, Nature Genetics 215 Mutations Identified in Exome and TCA No Prognostic Significance for Initiating Ras pathway alteration Stieglitz, Taylor Wiener, Nature Genetics 215 Stieglitz, Taylor Wiener, Nature Genetics 215 More Alterations Confer Worse Outcome PTPN11 PTPN11 + SH2B3 Stieglitz, Taylor Wiener, Nature Genetics 215 8

9 Summary Acknowledgements JMML is a genetically more complex disease than previously documented, with implications for mono and combinatorial therapies (?will MEK inhibition work?) Documentation of multiple genetic events can clarify diagnosis and identify patients at highest risk of relapse Discovery of additional genetic events informs future mechanistic studies of how Ras signaling is affected by protein X, Y, Z. Elliot Stieglitz Ernesto Diaz Flores Laura Gelston Thai Tran Tiffany Chang Kyle Beckman Kailyn Kim Emilio Esquivel Sophie Archambeault Michelle Kang Debbie Sakai Kevin Shannon Ben Braun Amaro Taylor Wiener Todd Golub Kim Stegmaier Michael Wang Scott Olsen Adam Abate John Haliburton Future Directions Deep Sequencing Misses Subclonal Mutations Ras Pathway Mutations SETBP1 Mutation Does SETBP1 Occur in a Progenitor or Initiating Cell? Gene Nucleotide Amino Acid Allelic Fraction Nucleotide Amino Acid Allelic Fraction J259 NRAS c.35g>t p.g12v.38 c.262g>a p.d868n.47 J264 PTPN11 c.226g>a p.e76k.21 c.269g>a p.g87d <.2* J276 NF1 c.1722c>g p.s574r.55 c.262g>a p.d868n <.2* J286 PTPN11 c.182a>t p.d61v.57 c.269g>a p.g87d <.2* J295 PTPN11 c.226g>a p.e76k.46 c.262g>a p.d868n <.2* J313 NRAS c.38g>a p.g13d.28 c.262g>a p.d868n.38 J322 PTPN11 c.182a>t p.d61v.52 c.262g>a p.d868n <.2* J342 PTPN11 c.226g>a p.e76k.48 c.262g>a p.d868n <.2* J c.262g>a p.d868n <.2* J43 NRAS c.38g>a p.g13d.21 c.262g>a p.d868n <.2* J44 CBL c.1112a>c p.y371s.82 c.269g>a p.g87d <.2* NRAS c.35g>a p.g12d.7 Modified from Sakaguchi et al. (213) J45 NRAS c.38g>a p.g13d.36 c.262g>a p.d868n <.2* 9

10 SETBP1 Affects Putative Stem Cells Human HSCs (Lin CD34 + CD38 + CD45RA CD9 + ) Laura Gelston Jon Akutagawa 1