Work in the Green lab is supported by
Leukemia and Lymphoma Research, Cancer Research UK, the NIHR Cambridge
Biomedical Research Centre, the Cambridge Experimental Cancer Medicine Centre,
and the Leukemia and Lymphoma Society of America. WW is supported by the
Austrian Science Foundation (J 3578-B21). JN is supported by a Kay Kendall
Leukaemia Clinical Fellowship.This is the final published version. It first appeared at http://www.nature.com/leu/journal/vaop/ncurrent/full/leu2014285a.html

A Tefferi

Claire N. Harrison

E Puigdecanet

F Passamonti

H Yoshida

J Nangalia

John T. Reilly

M Michalak

M Wictome

P Guglielmelli

R Rampal

S Schwemmers

S Verstovsek

T Klampfl

K Kollmann

W Warsch

H Quentmeier

A Bench

E Boyd

M Scott

H G Drexler

A R Green

Crossref

Leukemia

MARIMO cells harbor a CALR mutation but are not dependent on JAK2/STAT5 signaling

Apollo (Cambridge)

OPENLETTER TO THE EDITORMARIMO cells harbor a CALR mutation but are notdependent on JAK2/STAT5 signalingLeukemia advance online publication, 17 October 2014; doi:10.1038/leu.2014.285Mutations in calreticulin (CALR) were recently describedto be present in the majority of patients with a JAK2-unmutated myeloproliferative neoplasm (MPN).1,2 Thisdiscovery has had rapid clinical impact, and testing forCALR has been embedded in national and internationaldiagnostic guidelines.3–5 However, a human MPN-derived cellline harboring a CALR mutation has not been reported and themechanisms by which mutated-CALR results in an MPN remainunclear.To begin to investigate the pathogenetic consequences ofmutant CALR, we searched for patient-derived cell linesharboring CALR mutations. None were identified by exomesequencing of 1015 cell lines, including 37 derived fromhematopoietic neoplasms.1 We therefore looked for cell linesderived from patients with leukemic transformation of apreceding MPN. Given that CALR and JAK2 mutations arealmost completely mutually exclusive,1,2,6 we focused on foursuch lines known to lack a JAK2 mutation (MONO-MAC-6,MARIMO, GDM-1 and ELF-153), and also tested a further 52 otherpredominantly myeloid cell lines (Supplementary Table 1).Mutation screening was done by Sanger sequencing aspreviously described,1 and details of other methods are in theSupplementary Information.The only cell line found to harbor a CALR mutation wasMARIMO, originally derived from a 68-year-old female withAML-M2, and an antecedent history of ET.7 MARIMO is negativefor JAK2V617F and MPL exon 10 mutations (data not shown)and carries a heterozygous 61-basepair (bp) deletion in CALRexon 9 (c.1099_1159del; L367fs*43), which, like all otherreported CALR mutations, results in a +1- bp shift in the readingframe and thus generates a novel C terminus (Figure 1a). Inpatients, the commonest two CALR mutations, accounting for85% of cases, are a 52-bp deletion (type 1; c.1099_1150del;L367fs*46) and a 5-bp insertion (type 2; c.1154_1155_ins;K385fs*47).8 Both the type 1 deletion and the MARIMO deletionare immediately preceded by a nucleotide sequence identicalto that at the 3’ end of the deletion (Figure 1a). The 61-bpMARIMO deletion is readily detected by fragment analysis andrepresents a useful positive control for diagnostic clinical testing(Figure 1b).Allele-specific PCR demonstrated expression of the mutant CALRallele (Figure 1c). Compared with other cell lines derived frompatients with JAK2V617F (HEL, UKE-1 and SET-2) or CML (K562)total CALRmRNA levels were 10-fold higher in MARIMO (Figure 1d)and total CALR protein levels were also increased albeit moremodestly (Figure 1e).MARIMO cells expressed cell surface marker CD15 but not otherprogenitor or lineage-affiliated markers (Supplementary Table 2).The proliferation and cell cycle status of MARIMO was unremark-able compared with other myeloid lines (Supplementary Figure 1).We next analysed cellular calcium stores since CALR has animportant role in endoplasmic reticulum (ER) mediated calciumhomeostasis9 and mutant CALR protein lacks variable numbersof calcium binding sites present in the wild-type C terminus.No significant differences in basal cytoplasmic calcium levelswere found amongst the six cell lines tested (Figure 2a).Cell lines were then treated with 1 μM thapsigargin, which blocksER Ca2+-ATPase channels resulting in ER calcium depletion andincreased cytosolic calcium levels.10 MARIMO cells showed theslowest rate of increase of cytoplasmic calcium levels uponaddition of thapsigargin (Figure 2b), consistent with the conceptthat mutant CALR alters ER dependent calcium homeostasis.The mutual exclusivity of JAK2 and CALR mutationsargues that they may share pathogenetic mechanisms and hasbeen used to suggest that CALR mutations may activateJAK2/STAT5 signaling. This concept is supported by expressionprofiling of patient-derived granulocytes11 together with areport that expression of CALR in Ba/F3 cells confersinterleukin-3 independence and is accompanied by increasedSTAT5 phosphorylation.2 However other studies have reporteddistinct transcriptional signatures in JAK2V617F-mutated andJAK2V617F-unmutated MPNs.12,13 Interpretation of theseapparently conflicting results is complicated by several issuesincluding limitations of overexpression systems, the uncertainrelevance of granulocytes to disease pathogenesis anddifficulties inherent to studies of signaling in primarycells containing variable proportions of mutant cells. Tocircumvent some of these issues, and to gain insight into theconsequences of CALR mutations, we explored the properties ofMARIMO cells.The dependence of MARIMO cells on JAK signalingwas initially assessed using the JAK inhibitors Tofacitinib(a JAK2/3 inhibitor) and JAK-inhibitor-I (a pan-JAK inhibitor)(Supplementary Figure 2). MARIMO cells were more resistant toboth inhibitors than seven cell lines harboring mutantJAK2 or JAK3. Dose response studies using the clinicallyapproved JAK-inhibitor Ruxolitinib (INCB018424, a JAK1/2inhibitor) showed that HEL and UKE-1 (both JAK2V617F positive)had IC50 values of 217 and 430 nM, respectively (Figure 2c).In marked contrast the IC50 value for MARIMO was greater than10 000 nM, demonstrating that MARIMO was not dependent onJAK2 signaling. Consistent with these data, western blot analysisshowed that, compared with JAK2-mutant cells, MARIMO cellscontained markedly reduced levels of JAK2, phosphorylated-JAK2 (pJAK2), STAT5 and pSTAT5 (Figure 2d). The lack of JAK2-STAT5 signaling was not accompanied by a compensatoryincrease in STAT1 or STAT3 phosphorylation (Figure 2e). JAK2transcript levels in MARIMO were similar to other cell lines(Figure 2f), suggesting either decreased translation or increaseddegradation of JAK2.Together, our data demonstrate that the MARIMO cell lineharbors a CALR mutation and yet is not dependent on JAK/STATsignaling, in marked contrast to JAK2-mutated cell lines.Our results therefore raise the possibility that mutations ofCALR and JAK2 may share activation of pathways other than theSTATs. Superficially our data appear to contrast with reports thatJAK2-unmutated and CALR-mutated MF patients respond toAccepted article preview online 24 September 2014Leukemia (2014), 1–4© 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14www.nature.com/leuruxolitinib.14,15 However in JAK2V617F-positive patients studiesof mutant allele burden show that Ruxolitinib has a minimaleffect on the mutant clone.15 It is therefore likely that the clinicalresponses to Ruxolitinib (reduced splenomegaly and improvedconstitutional symptoms) do not reflect a cytotoxic effectof the drug on the neoplastic clone, but instead are at least inTotal CALR mRNATotal CALRHSC-70MARIMOK562HELUKE-1SET-2Total CALR proteinMARIMO: 61bp deletion (L367fs*43)Type 1: 52bp deletion (L367fs*46)CALR exon 95 3Type 2: 5bp insertion (K385fs*47)TTGTC417411408430AAWild typeType 1: L367fs*46Type 2: K385fs*47MARIMO: L367fs*43Amino acid sequenceNCwt61bpdeletionCALRDye signalintensity Fragment size (bp)61bp deletion (MARIMO; L367fs*43) 52bp deletion (Type 1; L367fs*46) 5bp insertion (Type 2; K385fs*47)132bp(wt)132bp(wt)132bp(wt)fold change mRNA 051015***MARIMOMARIMOFigure 1. Identification of a CALR-mutated human cell line. (a) Top panel shows the mutated region in CALR exon 9 (red bases). Thecommonest CALR mutations are shown above the DNA sequence. Solid gray line shows type 1 (52- bp deletion; c.1099_1150del; L367fs*46)and gray arrow shows type 2 (5- bp insertion; c.1154_1155_ins; K385fs*47 mutations). The CALRmutation in human cell line MARIMO is shownbelow the DNA sequence. Solid red line and capillary sequencing image show a heterozygous 61-bp deletion (c.1099_1159del; L367fs*43) inMARIMO. Dashed gray and red lines represent the homologous sequence flanking the deleted regions in type 1 and MARIMO mutations,respectively, also highlighted in the capillary sequencing image (pale blue) for MARIMO. Lower panel shows the predicted protein sequenceof the commonest CALR mutations and of MARIMO with total protein sizes. Amino acids (AA) in the new reading frame are shaded blue andthe common novel peptide sequence shared by the different CALR variants are in bold blue. (b) PCR amplification of CALR exon 9 followed byfragment size analysis, as used for diagnostic testing for CALR mutations. Vertical heights of peaks represent dye signal intensity andhorizontal position of peaks reflect the fragment size of the PCR amplicon. Wild type (wt) peak occurs at 132- bp. Left panel shows wt andmutated alleles of MARIMO (61-bp separation in peaks), middle panel shows Type 1/L367fs*46 with peak separation of 52 bp and right panelshows Type 2/K385fs*47 peaks separated by 5 bp. (c) Agarose gel image showing wt (upper band) and mutated-CALR (lower band) inMARIMO DNA and cDNA. (d) Quantitative real-time PCR of total CALRmRNA levels expressed as a fold change relative to house-keeping RPLP0levels, for the cell lines MARIMO, the BCR-ABL1+ CML cell line K562, and the JAK2V617F+ cell lines HEL, UKE-1 and SET-2. Graph depicts all datapoints generated in two independent experiments performed in duplicate. ***Po0.001 (e) Western blot showing total CALR protein levels ofMARIMO and four other myeloid cell lines.Letter to the Editor2Leukemia (2014) 1 – 4 © 2014 Macmillan Publishers Limitedpart due to down-modulation of pro-inflammatory signalingcascades.16CONFLICT OF INTERESTThe authors declare no conflict of interest.ACKNOWLEDGEMENTSWe are grateful to Hitoshi Kiyoi (Nagoya University) for provision of cell lineMARIMO. We thank Barbara Newman, Haemato-Oncology Diagostic Service,Addenbrooke's Hospital, Cambridge, UK. Work in the Green lab is supported byLeukemia and Lymphoma Research, Cancer Research UK, the NIHR CambridgeBiomedical Research Centre, the Cambridge Experimental Cancer Medicine Centre,and the Leukemia and Lymphoma Society of America. WW is supported by theAustrian Science Foundation (J 3578-B21). JN is supported by a Kay KendallLeukaemia Clinical Fellowship.AUTHOR CONTRIBUTIONSKK performed western blots and reverse transcriptase–PCR; WW performed calciumrelease-, inhibitor-, proliferation- and cell cycle assays; JN performed cell line screen;HQ and HD prepared cell lines; AB, EB performed clinical assays. All authors wrote andreviewed the manuscript. ARG directed the research.150 300 450 600 900Calcium dye MFI(Δ basal calcium level)Thapsigargin addedpSTAT5STAT5pJAK2JAK2HSC-70MARIMOK562HELUKE-1SET-2pSTAT1pSTAT3STAT3STAT1HSC-70MARIMOK562HELUKE-1SET-2PC0.00.51.01.52.0fold change JAK2 mRNA020040060080010001200Basal calcium dye MFI HEL 217UKE-1 430K562 > 10,000MARIMO > 10,000% living cellsIC50 [nM]Ruxolitinib [nM]Ruxolitinib0 102 103 10402040608010012005001000150020002500HELMARIMOK562UKE-1MOLM13HL-60Time [sec]Figure 2. Characterization of the cell line MARIMO. (a and b) Basal cytoplasmic calcium level (a) and changes in cytoplasmic calcium levelsover time upon addition of thapsigargin (b) in MARIMO and five other leukemic cell lines. (c) Dose response curves for the JAK2-inhibitorRuxolitinib in the cell lines MARIMO, K562, HEL and UKE-1. (d and e) Western blots showing protein levels of the inactive and phosphorylatedforms of JAK2 and STAT5 (d) and STAT1 and STAT3 (e). PC, positive control. (f) JAK2 mRNA levels expressed as fold changes relative to RPLP0 inMARIMO and four myeloid cell lines.Letter to the Editor3© 2014 Macmillan Publishers Limited Leukemia (2014) 1 – 4K Kollmann1,5, J Nangalia1,2,3,5, W Warsch1,5, H Quentmeier4,A Bench3, E Boyd3, M Scott3, HG Drexler4 and AR Green1,31Cambridge Institute for Medical Research, Wellcome Trust/MRCStem Cell Institute and Department of Haematology, University ofCambridge, Cambridge, UK;2Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK;3Cambridge University Hospitals NHS Foundation Trust,Cambridge, UK and4Leibniz-Institute DSMZ, German Collection of Microorganisms andCell Cultures, Braunschweig, GermanyE-mail: ww305@cam.ac.uk or arg1000@cam.ac.uk5These authors contributed equally to this workREFERENCES1 Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC et al. SomaticCALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl JMed 2013; 369: 2391–2405.2 Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD et al.Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med2013; 369: 2379–2390.3 Tefferi A, Thiele J, Vannucchi AM, Barbui T. An overview on CALR and CSF3Rmutations and a proposal for revision of WHO diagnostic criteria for myelopro-liferative neoplasms. Leukemia 2014; 28: 1407–1413.4 Reilly JT, McMullin MF, Beer PA, Butt N, Conneally E, Duncombe AS et al. Use ofJAK inhibitors in the management of myelofibrosis: a revision of the BritishCommittee for Standards in Haematology Guidelines for Investigation andManagement of Myelofibrosis 2012. Br J Haematol 2014; e-pub ahead of print25 June 2014; doi:10.1111/bjh.12985.5 Harrison CN, Butt N, Campbell P, Conneally E, Drummond M, Green AR et al.Modification of British Committee for Standards in Haematology diagnosticcriteria for essential thrombocythaemia. Br J Haematol 2014; e-pub ahead of print17 June 2014; doi:10.1111/bjh.12986.6 Tefferi A, Lasho TL, Finke CM, Knudson RA, Ketterling R, Hanson CH et al.CALR vs JAK2 vs MPL-mutated or triple-negative myelofibrosis: clinical,cytogenetic and molecular comparisons. Leukemia 2014; 28: 1472–1477.7 Yoshida H, Kondo M, Ichihashi T, Hashimoto N, Inazawa J, Ohno R et al. A novelmyeloid cell line, Marimo, derived from therapy-related acute myeloid leukemiaduring treatment of essential thrombocythemia: consistent chromosomalabnormalities and temporary C-MYC gene amplification. Cancer Genet Cytogenet1998; 100: 21–24.8 Guglielmelli P, Nangalia J, Green AR, Vannucchi AM. CALR mutations inmyeloproliferative neoplasms: hidden behind the reticulum. Am J Hematol 2014;89: 453–456.9 Michalak M, Groenendyk J, Szabo E, Gold LI, Opas M. Calreticulin, a multi-processcalcium-buffering chaperone of the endoplasmic reticulum. Biochem J 2009; 417:651–666.10 Wictome M, Henderson I, Lee AG, East JM. Mechanism of inhibition of thecalcium pump of sarcoplasmic reticulum by thapsigargin. Biochem J 1992; 283:525–529.11 Rampal R, Al-Shahrour F, Abdel-Wahab O, Patel JP, Brunel J-P, Mermel CH et al.Integrated genomic analysis illustrates the central role of JAK-STAT pathwayactivation in myeloproliferative neoplasm pathogenesis. Blood 2014; 123:e123–e133.12 Schwemmers S, Will B, Waller CF, Abdulkarim K, Johansson P, Andreasson B et al.JAK2V617F-negative ET patients do not display constitutively active JAK/STATsignaling. Exp Hematol 2007; 35: 1695–1703.13 Puigdecanet E, Espinet B, Lozano JJ, Sumoy L, Bellosillo B, Arenillas L et al.Gene expression profiling distinguishes JAK2V617F-negative fromJAK2V617F-positive patients in essential thrombocythemia. Leukemia 2008; 22:1368–1376.14 Passamonti F, Caramazza D, Maffioli M. JAK inhibitor in CALR-mutant myelofi-brosis. N Engl J Med 2014; 370: 1168–1169.15 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; 366:799–807.16 Verstovsek S, Kantarjian H, Mesa RA, Pardanani AD, Cortes-Franco J, Thomas DAet al. Safety and efficacy of INCB018424, a JAK1 and JAK2 Inhibitor, in myelofi-brosis. N Engl J Med 2010; 363: 1117–1127.This work is licensed under a Creative Commons Attribution 4.0International License. The images or other third party material in thisarticle are included in the article’s Creative Commons license, unless indicatedotherwise in the credit line; if the material is not included under the CreativeCommons license, users will need to obtain permission from the license holder toreproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)Letter to the Editor4Leukemia (2014) 1 – 4 © 2014 Macmillan Publishers Limited

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MARIMO cells harbor a CALR mutation but are not dependent on JAK2/STAT5 signaling.

MARIMO cells harbor a CALR mutation but are not dependent on JAK2/STAT5 signaling.

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