Risk Factors and Mechanisms for the Initiation and ...

Risk Factors and Mechanisms for the Initiation and Progression of Myeloproliferative Neoplasms:

Will a better understanding yield an Achilles Heel or Penrose Stairs?

Andrew Kuykendall, MDAssistant MemberMoffitt Cancer Center

@KuykendallMD

• Disease Background• MPNs as a Model of Inflammation• MPN Development• MPN Progression• Future Considerations

Outline

MPN Overview

MPN Epidemiology

1. Anderson, L. A., & McMullin, M. F. (2014). Epidemiology of MPN: what do we know? Curr Hematol Malig Rep, 9(4), 340-349. doi:10.1007/s11899-014-0228-z2. Rollison, D. E., Howlader, N., Smith, M. T., Strom, S. S., Merritt, W. D., Ries, L. A., . . . List, A. F. (2008). Epidemiology of myelodysplastic syndromes and chronic myeloproliferative disorders in the United States,

2001-2004, using data from the NAACCR and SEER programs. Blood, 112(1), 45-52. doi:10.1182/blood-2008-01-134858

MPNs are associated with a diverse array of symptoms

Emanuel, R. M., Dueck, A. C., Geyer, H. L., Kiladjian, J. J., Slot, S., Zweegman, S., . . . Mesa, R. A. (2012). Myeloproliferative neoplasm (MPN) symptom assessment form total symptom score: prospective international assessment of an abbreviated symptom burden scoring system among patients with MPNs. J Clin Oncol, 30(33), 4098-4103. doi:10.1200/JCO.2012.42.3863

MPNs are associated with worse survival than age-matched controls

Price, G. L., Davis, K. L., Karve, S., Pohl, G., & Walgren, R. A. (2014). Survival patterns in United States (US) medicare enrollees with non-CML myeloproliferative neoplasms (MPN). PLoS One, 9(3), e90299. doi:10.1371/journal.pone.0090299

MPNs are driven by a mutation in JAK2, CALR, or MPL

Grinfeld, J., Nangalia, J., & Green, A. R. (2017). Molecular determinants of pathogenesis and clinical phenotype in myeloproliferative neoplasms. Haematologica, 102(1), 7-17. doi:10.3324/haematol.2014.113845

There is a familial risk with MPNs that is not linked to driver mutation

1. Landgren, O., Goldin, L. R., Kristinsson, S. Y., Helgadottir, E. A., Samuelsson, J., & Bjorkholm, M. (2008). Increased risks of polycythemia vera, essential thrombocythemia, and myelofibrosis among 24,577 first-degree relatives of 11,039 patients with myeloproliferative neoplasms in Sweden. Blood, 112(6), 2199-2204. doi:10.1182/blood-2008-03-143602

2. Rumi, E., & Cazzola, M. (2017). Diagnosis, risk stratification, and response evaluation in classical myeloproliferative neoplasms. Blood, 129(6), 680-692. doi:10.1182/blood-2016-10-6959573. Sud, A., Chattopadhyay, S., Thomsen, H., Sundquist, K., Sundquist, J., Houlston, R. S., & Hemminki, K. (2018). Familial risks of acute myeloid leukemia, myelodysplastic syndromes, and myeloproliferative

neoplasms. Blood, 132(9), 973-976. doi:10.1182/blood-2018-06-858597

MPNs are driven by a mutation in JAK2, CALR, or MPL

Nangalia, J., & Green, A. R. (2017). Myeloproliferative neoplasms: from origins to outcomes. Blood, 130(23), 2475-2483. doi:10.1182/blood-2017-06-782037

The hallmark of MPN is hyperactive JAK/STAT signaling

Rampal, R., Al-Shahrour, F., Abdel-Wahab, O., Patel, J. P., Brunel, J. P., Mermel, C. H., . . . Levine, R. L. (2014). Integrated genomic analysis illustrates the central role of JAK-STAT pathway activation in myeloproliferative neoplasm pathogenesis. Blood, 123(22), e123-133. doi:10.1182/blood-2014-02-554634

Additional inflammatory pathways beyond JAK/STAT are activated in MPNs

1. Fisher, D. A. C., Malkova, O., Engle, E. K., Miner, C. A., Fulbright, M. C., Behbehani, G. K., . . . Oh, S. T. (2017). Mass cytometry analysis reveals hyperactive NF Kappa B signaling in myelofibrosis and secondary acute myeloid leukemia. Leukemia, 31(9), 1962-1974. doi:10.1038/leu.2016.3772. Fisher, D. A. C., Miner, C. A., Engle, E. K., Hu, H., Collins, T. B., Zhou, A., . . . Oh, S. T. (2019). Cytokine production in myelofibrosis exhibits differential responsiveness to JAK-STAT, MAP kinase, and NFkappaB signaling. Leukemia, 33(8), 1978-1995. doi:10.1038/s41375-019-0379-y

Numerous inflammatory cytokines are increased in PMF

Tefferi, A., Vaidya, R., Caramazza, D., Finke, C., Lasho, T., & Pardanani, A. (2011). Circulating interleukin (IL)-8, IL-2R, IL-12, and IL-15 levels are independently prognostic in primary myelofibrosis: a comprehensive cytokine profiling study. J Clin Oncol, 29(10), 1356-1363. doi:10.1200/JCO.2010.32.9490

Specific Cytokines are Prognostic

Tefferi, A., Vaidya, R., Caramazza, D., Finke, C., Lasho, T., & Pardanani, A. (2011). Circulating interleukin (IL)-8, IL-2R, IL-12, and IL-15 levels are independently prognostic in primary myelofibrosis: a comprehensive cytokine profiling study. J Clin Oncol, 29(10), 1356-1363. doi:10.1200/JCO.2010.32.9490

General Inflammatory Markers are Prognostic

1. Barbui T, Carobbio A, Finazzi G, et al: Elevated C-reactive protein is associated with shortened leukemia-free survival in patients with myelofibrosis. Leukemia 27:2084-6, 20132. Barosi G, Massa M, Campanelli R, et al: Primary myelofibrosis: Older age and high JAK2V617F allele burden are associated with elevated plasma high-sensitivity C-reactive protein levels and a phenotype of progressive disease. Leuk Res 60:18-23, 2017

Numerous Cytokines are Implicated in MPN Progression

Longhitano L, Li Volti G, Giallongo C, et al: The Role of Inflammation and Inflammasome in Myeloproliferative Disease. J Clin Med 9, 2020

Ruxolitinib, a JAK1/2 inhibitor, decreases several cytokine levels

Verstovsek, S., Kantarjian, H., Mesa, R. A., Pardanani, A. D., Cortes-Franco, J., Thomas, D. A., . . . Tefferi, A. (2010). Safety and efficacy of INCB018424, a JAK1 and JAK2 inhibitor, in myelofibrosis. N Engl J Med, 363(12), 1117-1127. doi:10.1056/NEJMoa1002028

Ruxolitinib improves spleen size, symptom score, albumin level and survival in higher risk MF patients

1. Verstovsek, S., Mesa, R. A., Gotlib, J., Levy, R. S., Gupta, V., DiPersio, J. F., . . . Kantarjian, H. M. (2012). A double-blind, placebo-controlled trial of ruxolitinib for myelofibrosis. N Engl J Med, 366(9), 799-807. doi:10.1056/NEJMoa11105572. Mesa, R. A., Verstovsek, S., Gupta, V., Mascarenhas, J. O., Atallah, E., Burn, T., . . . Gotlib, J. (2015). Effects of ruxolitinib treatment on metabolic and nutritional parameters in patients with myelofibrosis from COMFORT-I. Clin Lymphoma Myeloma Leuk, 15(4), 214-221 e211.

doi:10 1016/j clml 2014 12 008

Spleen Symptoms

Albumin Survival

Some MF-upregulated cytokines are not reduced with ruxolitinib

1. Fisher, D. A. C., Miner, C. A., Engle, E. K., Hu, H., Collins, T. B., Zhou, A., . . . Oh, S. T. (2019). Cytokine production in myelofibrosis exhibits differential responsiveness to JAK-STAT, MAP kinase, and NFkappaB signaling. Leukemia, 33(8), 1978-1995. doi:10.1038/s41375-019-0379-

Ruxolitinib has modest impact on mutant allele burden

Deininger, M., Radich, J., Burn, T. C., Huber, R., Paranagama, D., & Verstovsek, S. (2015). The effect of long-term ruxolitinib treatment on JAK2p.V617F allele burden in patients with myelofibrosis. Blood, 126(13), 1551-1554. doi:10.1182/blood-2015-03-635235

So, what do we know?

• MPNs are rare, and their incidence increases with advancing age.

• There is a familial susceptibility to MPNs that is not linked to driver mutation.

• MPNs are driven by phenotype-driving mutations in one of three genes that all lead to activated JAK/STAT signaling; however, other inflammatory pathways are upregulated as well.

• The inflammatory cytokines upregulated in MPNs are linked to disease progression and inferior outcomes.

• Some of this inflammation is mitigated with blocking the JAK/STAT pathway and correlates with symptomatic improvement and prolonged survival, but modest disease modification.

So, what do we need to know?

• MPNs are rare, and their incidence increases with advancing age.• What factors are associated with an increased incidence of MPN?

• There is a familial susceptibility to MPNs that is not linked to driver mutation.• What accounts for this susceptibility?

• MPNs are driven by phenotype-driving mutation in one of three genes that activates JAK/STAT signaling; however, other inflammatory pathways are upregulated as well.

• What is the incidence of these mutations in the general (healthy) population?

• The inflammatory cytokines upregulated in MPNs are linked to disease progression and inferior outcomes.

• What is the role for these cytokines? Are some critical for disease initiation/progression vs mediating clinical symptoms?

• Some of this inflammation is mitigated with blocking the JAK/STAT pathway and correlates with symptomatic improvement and prolonged survival, but modest disease modification.

• Can blocking additional inflammatory pathways induce superior symptomatic improvement?

• Can targeting specific cytokines not inhibited by JAK inhibition lead to disease modification?

JAK2 clonal hematopoiesis is not uncommon and is associated with increased risk of cardiovascular disease

Jaiswal S, Natarajan P, Silver AJ, et al. Clonal Hematopoiesis and Risk of Atherosclerotic Cardiovascular Disease. N Engl J Med. 2017;377(2):111-121.

JAK2 mutations occur in healthy patients and their presence and burden correlates with increasing age.

Cordua S, Kjaer L, Skov V, et al: Prevalence and phenotypes of JAK2 V617F and calreticulin mutations in a Danish general population. Blood 134:469-479, 2019

Population Presence of JAK2mutations: 3.2%

What determines whether a JAK2 mutation

turns into an MPN?

?

Inflammation supports MPN Development

Fleischman AG: Inflammation as a Driver of Clonal Evolution in Myeloproliferative Neoplasm. Mediators Inflamm 2015:606819, 2015Fleischman AG, Aichberger KJ, Luty SB, et al: TNFalpha facilitates clonal expansion of JAK2V617F positive cells in myeloproliferative neoplasms. Blood 118:6392-8, 2011

Chronic Inflammation is Mediated by Cytokines and Fosters MPN Progression

Hasselbalch HC: The role of cytokines in the initiation and progression of myelofibrosis. Cytokine Growth Factor Rev 24:133-45, 2013

MPN is driven by inflammation

Andersen M, Sajid Z, Pedersen RK, et al: Mathematical modelling as a proof of concept for MPNs as a human inflammation model for cancer development. PLoS One 12:e0183620, 2017

MPN Development Can be Mathematically Modeled


Modulation of Inflammation Alters the MPN Timeline


Inflammation supports MPN Development

Craver, B. M., El Alaoui, K., Scherber, R. M., & Fleischman, A. G. (2018). The Critical Role of Inflammation in the Pathogenesis and Progression of Myeloid Malignancies. Cancers (Basel), 10(4). doi:10.3390/cancers10040104

What specific inflammatory pressures promote MPN formation?

• Inflammatory Bowel Disease• Obesity• Smoking• Polymorphisms Altering Inflammatory Balance

• miR-146a• IL-6R polymorphisms

• TNFα / IL-10 axis

There is an increased incidence of inflammatory bowel disease both before and after an MPN diagnosis

1. Bak M, Jess T, Flachs EM, Zwisler AD, Juel K, Frederiksen H. Risk of Inflammatory Bowel Disease in Patients with Chronic Myeloproliferative Neoplasms: A Danish Nationwide Cohort Study. Cancers (Basel). 2020;12(9).

2.4-fold increased risk of developing IBD after MPN diagnosis

40% increased risk of preceding IBD diagnosis prior to MPN

Smoking is associated with MPN Development

Jayasuriya NA, Kjaergaard AD, Pedersen KM, et al. Smoking, blood cells and myeloproliferative neoplasms: meta-analysis and Mendelian randomization of 2.3 million people. Br J Haematol. 2020;189(2):323-334.

Adolescent obesity may be related to increased incidence of MPNs

Leiba A, Duek A, Afek A, Derazne E, Leiba M. Obesity and related risk of myeloproliferative neoplasms among israeli adolescents. Obesity (Silver Spring). 2017;25(7):1187-1190.

miR-146a Expression Linked to Fibrotic Progression

Ferrer-Marin F, Arroyo AB, Bellosillo B, et al: miR-146a rs2431697 identifies myeloproliferative neoplasm patients with higher secondary myelofibrosis progression risk. Leukemia, 2020

• miR-146a is a brake in NF-kB signaling

• rs2431697 genotype is associated with lower levels of miR-146a leading to more active NF-kB activity

miR-146a is a significant brake on autoimmunity, myeloproliferation, and cancer in mice

Boldin, M. P., Taganov, K. D., Rao, D. S., Yang, L., Zhao, J. L., Kalwani, M., . . . Baltimore, D. (2011). miR-146a is a significant brake on autoimmunity, myeloproliferation, and cancer in mice. J Exp Med, 208(6), 1189-1201. doi:10.1084/jem.20101823

miR-146a knockout leads to dysregulated NF-kB and myeloproliferation

Zhou et al., NF-kB dysregulation in microRNA-146a-deficient mice drives the development of myeloid malignancies. Proc Natl Acad Sci U S A. 2011 May 31; 108(22):9184-9189

Loss of Function Polymorphism in IL6R Reduces Risk of MPN Formation

Pedersen KM, Colak Y, Ellervik C, et al: Loss-of-function polymorphism in IL6R reduces risk of JAK2V617F somatic mutation and myeloproliferative neoplasm: A Mendelian randomization study. EClinicalMedicine21:100280, 2020

Loss of Function Polymorphism in IL6R Reduces Risk of MPN Formation

Pedersen KM, Colak Y, Ellervik C, et al: Loss-of-function polymorphism in IL6R reduces risk of JAK2V617F somatic mutation and myeloproliferative neoplasm: A Mendelian randomization study. EClinicalMedicine 21:100280, 2020

Selective pressures may shape clonal expansion

Craver, B. M., El Alaoui, K., Scherber, R. M., & Fleischman, A. G. (2018). The Critical Role of Inflammation in the Pathogenesis and Progression of Myeloid Malignancies. Cancers (Basel), 10(4). doi:10.3390/cancers10040104

MPN monocytes produce more TNFα than normal monocytes

Lai, H. Y., Brooks, S. A., Craver, B. M., Morse, S. J., Nguyen, T. K., Haghighi, N., . . . Fleischman, A. G. (2019). Defective negative regulation of Toll-like receptor signaling leads to excessive TNF-alpha in myeloproliferative neoplasm. Blood Adv, 3(2), 122-131. doi:10.1182/bloodadvances.2018026450

Decreased response to IL-10 leads to high TNFαenvironment


Decreased response to IL-10 seen in affected and unaffected twin – suggesting an inherited susceptibility


IL-1β promotes disease initiation and progression

Rai, S., Hansen, N., Hao-Shen, H., Tata, N. R., & Skoda, R. C. (2019). IL-1B secreted from mutant cells carrying JAK2-V617F favors early clonal expansion and promotes MPN disease initiation and progression. Blood, 134 (Supplement_1), 307.

IL-1β Supports the Mutant HSC

1. Arranz L, Sanchez-Aguilera A, Martin-Perez D, et al: Neuropathy of haematopoietic stem cell niche is essential for myeloproliferative neoplasms. Nature 512:78-81, 20142. Sollazzo D, Forte D, Polverelli N, et al: Crucial factors of the inflammatory microenvironment (IL-1beta/TNF-alpha/TIMP-1) promote the maintenance of the malignant hemopoietic clone of myelofibrosis: an in vitro study. Oncotarget 7:43974-43988, 2016

IL-1β and TNFα support MPN HSC survival, enhance migratory behavior of MF CD34+ cells and increase clonogenic output

1. Arranz L, Sanchez-Aguilera A, Martin-Perez D, et al: Neuropathy of haematopoietic stem cell niche is essential for myeloproliferative neoplasms. Nature 512:78-81, 20142. Sollazzo D, Forte D, Polverelli N, et al: Crucial factors of the inflammatory microenvironment (IL-1beta/TNF-alpha/TIMP-1) promote the maintenance of the malignant hemopoietic clone of myelofibrosis: an in vitro study. Oncotarget 7:43974-43988, 2016

CXCL4 links inflammation and disease progression

Gleitz, H. F. E., Dugourd, A. J. F., Leimkuhler, N. B., Snoeren, I. A. M., Fuchs, S. N. R., Menzel, S., . . . Schneider, R. K. (2020). Increased CXCL4 expression in hematopoietic cells links inflammation and progression of bone marrow fibrosis in MPN. Blood, 136(18), 2051-2064. doi:10.1182/blood.2019004095

CXCL4 knockout in HSPCs ameliorates MPN phenotype


CXCL4 knockout in HSPCs ameliorates MPN phenotype


BET inhibition suppresses NF-kB-mediated inflammation and synergizes with JAK inhibition

Kleppe, M., Koche, R., Zou, L., van Galen, P., Hill, C. E., Dong, L., . . . Levine, R. L. (2018). Dual Targeting of Oncogenic Activation and Inflammatory Signaling Increases Therapeutic Efficacy in Myeloproliferative Neoplasms. Cancer Cell, 33(1), 29-43 e27. doi:10.1016/j.ccell.2017.11.009

Combined BET and JAK inhibition leads to spleen and symptom responses

Mascarenhas et al., CPI-0610, a Bromodomain and Extraterminal Domain Protein (BET) Inhibitor, in Combination with Ruxolitinib, in JAK-Inhibitor-Naïve Myelofibrosis Patients: Update of MANIFEST Phase 2 Study. Presented at ASH 2020.

Add-on of CPI-0610 leads to symptom benefit and transfusion independence

Mascarenhas et al., CPI-0610, a Bromodomain and Extraterminal Domain Protein (BET) Inhibitor, in Combination with Ruxolitinib, in JAK-Inhibitor-Naïve Myelofibrosis Patients: Update of MANIFEST Phase 2 Study. Presented at ASH 2020.

Pacritinib – a selective JAK2 inhibitor – also inhibits IRAK1 at nanomolar concentrations

Singer, J. W., Al-Fayoumi, S., Ma, H., Komrokji, R. S., Mesa, R., & Verstovsek, S. (2016). Comprehensive kinase profile of pacritinib, a nonmyelosuppressive Janus kinase 2 inhibitor. J Exp Pharmacol, 8, 11-19. doi:10.2147/JEP.S110702

Pacritinib is effective in reducing spleen volume in severely thrombocytopenic patients

Mesa RA, Vannucchi AM, Mead A, et al. Pacritinib versus best available therapy for the treatment of myelofibrosis irrespective of baseline cytopenias (PERSIST-1): an international, randomised, phase 3 trial. Lancet Haematol. 2017;4(5):e225-e236.

Summary

• MPNs are inflammatory diseases that are cultivated by and induce inflammation

• Numerous inflammatory pathways/mediators have been implicated in the pathogenesis of

MPNs

• Clinical targeting of these pathways has led to encouraging early clinical results

• Numerous trials are being developed to assess whether targeting the underlying inflammatory

pathways can modify the disease.

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