Reactive oxygen species overload promotes apoptosis in JAK2V617F-positive cell lines^☆

Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), including essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF), are characterized by excessive myeloid proliferation, with predominant megakaryocytic, erythroid, and megakaryocytic/granulocytic expansion, respectively, and have a potential of transformation to acute myeloid leukemia.¹ From the molecular point of view, the Janus kinase 2 (JAK2)/signal transducer and activator of transcription (STAT) signaling pathway plays an important role in the pathogenesis of MPNs. A recurrent gain-of-function mutation, V617F in JAK2, has been reported in most PV cases and in more than half of ET and PMF cases.² In JAK2V617F-negative patients, other gain-of-function mutations in genes related to JAK2/STAT signaling activation, including JAK2 exon 12, ³MPL⁴ and Calreticulin mutations, ^5and6 have been identified.

The current therapies for MPN are limited and do not result in the elimination of the malignant clone. The only curative approach for these diseases is allogeneic stem cell transplantation.⁷ Ruxolitinib is a selective JAK1/2 inhibitor approved by the Food and Drug Administration (FDA) of the United States for the treatment of intermediate and high-risk PMF and PV patients with inadequate response or intolerance to hydroxyurea. Results from phase III clinical trials demonstrated that ruxolitinib is well tolerated, reduces inflammatory cytokines and splenomegaly, and ameliorates constitutional symptoms in PMF patients.^8,9and10 Similarity, ruxolitinib controls the hematocrit levels, reduces the spleen volume, and improves symptoms in PV patients.¹¹ However, ruxolitinib treatment does not reverse bone marrow fibrosis, suggesting that additional therapeutic strategies are required.

Reactive oxygen species (ROS) play a singular role in MPN cell biology. Marty et al.¹² showed that hematopoietic cells from a JAK2V617F knock-in mice model present higher levels of ROS compared to those from normal mice, contributing to DNA damage and genomic instability, which promote disease progression. An increased basal level of ROS was also observed in primary hematopoietic cells from MPN patients compared to those from healthy donors.^13and14 Ahn et al., ¹⁴ exploring the molecular mechanism of elevated JAK2V167F-induced ROS levels and cell survival under this stress condition, found that, due to DNA damage, B-cell lymphoma-extra large (BCL-XL) repression may be compromised.

In the current edition of the Revista Brasileira de Hematologia e Hemoterapia, Tavares et al.¹⁵ report that l-amino acid oxidase (LAAO) derived from Calloselasma rhosostoma snake venom exhibits cytotoxicity and induces apoptosis in JAK2V617F-cell lines (HEL and SET2) in a ROS production-dependent manner. The anti-cancer effects of the LAAO isolated from the venom of other snake species has been described in solid tumors ^16,17and18 and leukemia ^19and20 cell lines. Notably, it has been reported that LAAO isolated from C. rhosostoma²⁰ and Bothrops pirajai¹⁹ did not exert a prominent cytotoxic effect in peripheral blood mononuclear cells isolated from healthy donors and that rusvinoxidase, the LAAO isolated from Daboia russelii russelii was non-toxic in mice, ²¹ suggesting that cancer cells may be more susceptible to the cytotoxicity induced by these compounds. Recently, Mukherjee et al.²¹ observed that treatment with rusvinoxidase induces ROS production and caspases activation, and also downregulates BCL-XL in the MCF-7 breast cancer cell line.

The work by Tavares et al.¹⁵ is an important step to establishing the cellular functions of LAAO in MPN cell models and provides additional insights into the development of new therapies. However, it is still necessary to establish the specific effects of LAAO isolated from C. rhosostoma in normal hematopoietic progenitors, primary cells from MPN patients and JAK2V617F-driven murine models. The research conducted by Tavares et al. ¹⁵ also paves the way to an important frontier of knowledge: even though JAK2V617F-positive cells exhibit increased ROS levels compared to normal cells, the overload of ROS can elicit apoptosis in JAK2V617F-positive cells. The better understanding of the molecular mechanisms involved in the survival of JAK2V617F-positive cells under oxidative stress may be an interesting therapeutic opportunity.

Based on the data presented by Tavares et al. in MPN models and the findings from other research groups using solid tumor models, and taking into account that JAK2/STAT5 activation leads to aberrant expressions of BCL-XL,²² future investigations verifying the effects of the combined treatment of JAK inhibitors and ROS inductors may be of interest.

References

• ¹
  Thoennissen NH, Krug UO, Lee DH, Kawamata N, Iwanski GB, Lasho T, et al. Prevalence and prognostic impact of allelic imbalances associated with leukemic transformation of Philadelphia chromosome- negative myeloproliferative neoplasms. Blood. 2010;115(14):2882-90.
• ²
  Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 2005;365(9464):1054-61.
• ³
  Scott LM, Tong W, Levine RL, Scott MA, Beer PA, Stratton MR, et al. JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis. N Engl J Med. 2007;356(5):
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  Pikman Y, Lee BH, Mercher T, McDowell E, Ebert BL, Gozo M, et al. MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia. PLoS Med. 2006;3(7):e270.
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  Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC, et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med. 2013;369(25):2391-405.
• ⁶
  Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD, et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med. 2013;369(25):2379-90.
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  Ballen KK, Shrestha S, Sobocinski KA, Zhang MJ, Bashey A, Bolwell BJ, et al. Outcome of transplantation for myelofibrosis. Biol Blood Marrow Transplant. 2010;16(3): 358-67.
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  Pardanani A, Vannucchi AM, Passamonti F, Cervantes F, Barbui T, Tefferi A. JAK inhibitor therapy for myelofibrosis: critical assessment of value and limitations. Leukemia. 2011;25(2):218-25.
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  Harrison C, Kiladjian JJ, Al-Ali HK, Gisslinger H, Waltzman R, Stalbovskaya V, et al. JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis. N Engl J Med. 2012;366(9):787-98.
• ¹⁰
  Verstovsek S, Mesa RA, Gotlib J, Levy RS, Gupta V, DiPersio JF, et al. A double- blind, placebo- controlled trial of ruxolitinib for myelofibrosis. N Engl J Med. 2012;366(9): 799-807.
• ¹¹
  Vannucchi AM, Kiladjian JJ, Griesshammer M, Masszi T, Durrant S, Passamonti F, et al. Ruxolitinib versus standard therapy for the treatment of polycythemia vera. N Engl J Med. 2015;372(5):426-35.
• ¹²
  Marty C, Lacout C, Droin N, Le Couedic JP, Ribrag V, Solary E, et al. A role for reactive oxygen species in JAK2 V617F myeloproliferative neoplasm progression. Leukemia. 2013;27(11):2187-95.
• ¹³
  Vener C, Novembrino C, Catena FB, Fracchiolla NS, Gianelli U, Savi F, et al. Oxidative stress is increased in primary and post-polycythemia vera myelofibrosis. Exp Hematol. 2010;38(11):1058-65.
• ¹⁴
  Ahn JS, Li J, Chen E, Kent DG, Park HJ, Green AR, et al. JAK2V617F mediates resistance to DNA damage-induced apoptosis by modulating FOXO3A localization and Bcl-xL deamidation. Oncogene. 2016;35(17):2235-46.
• ¹⁵
  Tavares CF, Maciel TF, Burin SM, Ambrósio L, Ghisla S, Sampaio SV, et al. L- Amino acid oxidase isolated from Calloselasma rhodostoma snake venom induces cytotoxicity and apoptosis in JAK2V617F-positive cell lines. Rev Bras Hematol Hemoter. 2016;38(2):128-34.
• ¹⁶
  Fung SY, Lee ML, Tan NH. Molecular mechanism of cell death induced by king cobra (Ophiophagus hannah) venom L- amino acid oxidase. Toxicon. 2015;96:38-45.
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  Lee ML, Fung SY, Chung I, Pailoor J, Cheah SH, Tan NH. King cobra (Ophiophagus hannah) venom L- amino acid oxidase induces apoptosis in PC-3 cells and suppresses PC-3 solid tumor growth in a tumor xenograft mouse model. Int J Med Sci. 2014;11(6):593-601.
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  Li Lee M, Chung I, Yee Fung S, Kanthimathi MS, Hong Tan N. Antiproliferative activity of king cobra (Ophiophagus hannah) venom L- amino acid oxidase. Basic Clin Pharmacol Toxicol. 2014;114(4):336-43.
• ¹⁹
  Burin SM, Ayres LR, Neves RP, Ambrosio L, de Morais FR, Dias-Baruffi M, et al. L- Amino acid oxidase isolated from Bothrops pirajai induces apoptosis in BCR-ABL-positive cells and potentiates imatinib mesylate effect. Basic Clin Pharmacol Toxicol. 2013;113(2):103-12.
• ²⁰
  Costa TR, Menaldo DL, Prinholato da Silva C, Sorrechia R, de Albuquerque S, Pietro RC, et al. Evaluating the microbicidal, antiparasitic and antitumor effects of CR- LAAO from Calloselasma rhodostoma venom. Int J Biol Macromol. 2015;80:489-97.
• ²¹
  Mukherjee AK, Saviola AJ, Burns PD, Mackessy SP. Apoptosis induction in human breast cancer (MCF-7) cells by a novel venom L- amino acid oxidase (Rusvinoxidase) is independent of its enzymatic activity and is accompanied by caspase-7 activation and reactive oxygen species production. Apoptosis. 2015;20(10):1358-72.
• ²²
  Garcon L, Rivat C, James C, Lacout C, Camara- Clayette V, Ugo V, et al. Constitutive activation of STAT5 and Bcl-xL overexpression can induce endogenous erythroid colony formation in human primary cells. Blood. 2006;108(5):1551-4.

• ☆
  See paper by Tavares et al. in Rev Bras Hematol Hemoter. 2016;38(2):123-34.

Publication Dates

• Publication in this collection
  Jul-Sep 2016