Genetic analysis and preclinical modeling of leukemic transformation of myeloproliferative neoplasms: Implications for therapeutic strategies

Transcription

1 Genetic analysis and preclinical modeling of leukemic transformation of myeloproliferative neoplasms: Implications for therapeutic strategies Raajit Rampal, MD, PhD Assistant Attending Physician Leukemia Service Memorial Sloan Kettering Cancer Center New York, NY

2 Disclosures for Raajit Rampal, MD, PhD Royalty Receipt of intellectual property/ Patent holder Consulting fee Speakers bureau Fees for non-cme services Contracted research Ownership interest (stocks, stock options) Other N/A N/A Incyte Corporation N/A Incyte Corporation N/A N/A N/A N/A = Not Applicable (no conflicts listed) Presentation includes discussion of off-label or unapproved use of a drug or medical device: N/A

3 Presentation and Diagnosis 72 y/o was referred for further management of primary myelofibrosis Patient found to have leukocytosis (30,000; normal Hgb and Plt count) incidentally during routine CBC in 2012 Bone marrow biopsy demonstrated hypercellular marrow with trilineage hematopoiesis and 2+ reticulin fibrosis. No increase in blasts noted No HSM on examination Molecular studies: negative for BCR-ABL and JAK2 mutation. Patient started on ASA 81mg and monitored expectantly.

4 Clinical History-1 Patient developed increasing fatigue, early satiety, night sweats, 20 lbs weight loss within 6 months of initial diagnosis Developed palpable splenomegaly WBC remained > 30,000, with presence of circulating blasts (3%-5%). Ruxolitinib started due to constitutional symptoms. Hgb and Plts begin to decline, but leukocytosis increases. Ruxolitinb discontinued and hydroxyurea started (1 year after initial diagnosis). Pancytopenia develops; hydroxyurea discontinued, but pancytopenia persists. Peripheral blast count increases to 5%-10%

5 Clinical History-2 Patient admited for low grade fevers and increasing leukocytosis (>75,000) Bone marrow: hypercellular marrow (100% cellularity) with 22% blasts. Cytogenetics monosomy 7 in 40% of metaphases Molecular Diagnostics: Presence of ASXL1 mutation (no JAK2 or MPL mutation identified; CALR not available).

6 Clinical History-3 Patient started on decitabine (10-day course) and completes 2 full cycles with persistence of circulating blasts and leukocytosis. Follow-up bone marrow demonstrates 58% blasts Patient switched to induction chemotherapy (daunorubicin and cytarabine). Day 14 bone marrow demonstrates 80% blasts Patient developed septic shock with MODS; transitioned to comfort care.

7 Poor Prognosis of Leukemic Transformation post-mpn -Need for better characterization of biological and genetic processes involved in transformation to AML -Need for genetically accurate models of leukemic transformation -Need for new therapeutic strategies Mesa RA et al. Blood 2005; 105:

8 Leukemic Transformation: Natural History Estimated risk of leukemic transformation (100 months from diagnosis) ET: 4% PV: 8.7% MF: 20.6% Cervantes F et al. Blood 2009;113: Cervantes F et al. Acta Haem 1991;85:124-7

9 Clinical Risk Factors for Leukemic Transformation Rampal and Mascarenhas Curr Opin Hematology 2014;21:65-71

10 Genetic Risk Factors for Leukemic Transformation Vannucchi AM et al. Leukemia 2013;27:1861-9

11 Implications of JAK2, MPL, and CALR Mutations Tefferi A et al. Leukemia 2014;28:1472-7

12 Clonal Evolution in MPN Lundberg P et al. Blood 2014;123:2220-8

13 Should We Test for More than JAK2/MPL/CALR? Pros: More potential information about prognosis Identification of new targets Opportunity to learn more about a patient s disease Cons: Unlikely to change management for most patients May be expensive May be anxiety provoking

14 Genetic Alterations in MPN Patients Role of different MPN disease alleles in disease initiation and maintenance is not fully delineated Mutations in epigenetic modifiers can contribute to chronic myeloproliferation or transformation to AML - >mechanisms not known Levine RL et al. Nature Rev Cancer 2007;7:

15 High-Throughput Mutational Profiling of Post-MPN AML DNA and RNA extracted, blood or bone marrow aspirates (BMA) of 33 patients Hybrid capture using custom baitsets targeting 374 cancer-related genes and for DNA-seq, and 272 genes frequently rearranged for RNA-seq. Enriched libraries were sequenced to high depth; DNA-seq libraries averaged >590 median exon coverage and RNA-seq libraries averaged >20M total pairs (49x49 paired-end reads)) Rampal RK et al. Blood 2013;122: abstr 4098

16 Frequency of Genomic Alterations in Post-MPN AML Rampal RK et al. Blood 2013;122: abstr 4098

17 Distinct Genetic Profiles of de novo and Post-MPN AML DNMT3A FLT3 De novo AML (ECOG 1900) Secondary AML from MPNs Patel JP et al. NEJM 2012;366: Rampal RK et al. Blood 2013;122: abstr 4098

18 NRAS Mutations Are Frequent Events in JAK2wt Post-MPN AML Rampal RK et al. Blood 2013;122: abstr 4098

19 CALR Mutations Occur Frequently in Post-MPN AML and Are Exclusive of JAK2 mutations Rampal R et al. PNAS, in press

20 SRSF2 Mutations Are Associated with Adverse Survival in Post-MPN AML Rampal R et al. PNAS, in press

21 JAK2, TET2, ASXL1 and Cytogenetic Alterations Do Not Significantly Impact Survival in Post-MPN AML Rampal R et al. PNAS, in press

22 TP53 Mutations Are Present in Dominant Clone in Post-MPN AML Rampal R et al. PNAS, in press

23 Murine Modeling of Leukemic Transformation Tp53 null mice Tp53 wildtype mice (control) Assays: -Survival -Blood counts -Pathology/Morphology --Serial Transplantability of Disease -Immunophenotyping Rampal R et al. PNAS, in press

24 Jak2V617F/Tp53 Null Mice Is Associated with Impaired Survival and Leukocytosis Rampal R et al. PNAS, in press

25 Jak2V617F/Tp53 Null Mice Develop Organomegaly Rampal R et al. PNAS, in press

26 Jak2V617F/Tp53 Null Mice Develop Acute Myeloid Leukemia Peripheral Blood Bone Marrow Cytospin Rampal R et al. PNAS, in press

27 Leukemic Cells from Jak2V617F/Tp53 Null Mice Show Increased Self-Renewal Rampal R et al. PNAS, in press Comparison Primary vs. secondary Primary vs. tertiary Tertiary vs. secondary P=value p<0.05 P<0.05 P<0.05

28 Spectrum of Anti-Leukemic Agents Demonstrate Efficacy in Murine Post-MPN AML INCB18424 PU-H71 CYT 387 LDE225 Rampal R et al. PNAS, in press

29 Spectrum of Anti-Leukemic Agents Demonstrate Efficacy in Murine Post-MPN AML Decitabine Decitabine/ INCB18424 Rampal R et al. PNAS, in press

30 HSP90 Inhibition as an Alternate Approach to Target JAK2 JAK2 is a HSP90 client protein, and that HSP90 inhibitors (including PU-H71) lead to JAK2 degradation (Marubayashi S et al. JCI 2010;120: )

31 In vivo Drug Trial in Secondary Transplant of Spleen-Derived Leukemic Cells 60 mg/kg INCB18424 oral gavage BID daily 2 million Splenocytes from Jak2V617F/Tp53-/- leukemic mouse PU-H71 75mg/kg IP M,W,F 0.5% Methycellulose (placebo) oral gavage BID daily Rampal R et al. PNAS, in press

32 INCB18424 and PU-H71 Prolong Survival in Jak2V617F/Tp53null Leukemic Mice Placebo vs PU-H71 P<0.05 Placebo vs INCB18424 P<0.05 INCB18424 vs PU-H71 P<0.05 *Log-Rank Test Rampal R et al. PNAS, in press

33 Treatment with PU-H71 or INCB18424 Reduces Spleen and Liver Weights Rampal R et al. PNAS, in press

34 INCB18424 and PU-H71 Increase Differentiation Placebo INCB18424 PU-H71 Rampal R et al. PNAS, in press

35 Conclusions Several clinical factors are associated with risk of leukemic transformation. Mutations in ASXL1, EZH2, IDH1/2, SRSF2 are associated with an increased risk of leukemic transformation The role of serial mutational analysis of patients is unclear Genomic studies reveal the mutational spectrum of post-mpn AML differs from de novo AML JAK2V617F + TP53 is a common mutational combination in post-mpn AML, and is sufficient to induce AML in vivo This novel model of post-mpn AML can be used to credential novel therapeutic approaches, many of which may be translated into clinical investigational strategies.

36 Acknowledgements Levine Lab Jihae Ahn Todd Hricik Sophie McKenney Jay Patel Alan Shih Priya Koppikar Neha Bhagwat Lindsay Saunders Olga Guryanova Ria Kleppe Franck Rapaport Kaitlyn Shank Matt Keller Sara Meyer MSKCC Leukemia Service Omar Abdel-Wahab Martin Tallman MSKCC Pathology Service Christopher Park Foundation Medicine Vince Miller Phil Stephens Doron Lipson MD Anderson Srdan Verstovsek Taghi Manshouri Mount Sinai John Mascarenhas Ronald Hoffman