Letters to the Editor 1759 Leukemic stem cells of patients carrying NPM1 are candidates for targeted immunotherapy

Leukemia (2014) 28, 1759–1762; doi:10.1038/leu.2014.116 prognostic and predictive maker.7 AML patients with normal karyotype carrying NPM1mut have a significant better overall survival compared with wild-type patients without FLT3-ITD.8 However, the underlying mechanisms are still not clear. These Targeting minimal residual disease (MRD) to prevent relapse is cause an abnormal shift of the NPM1 from the one of the major challenges in treatment of acute leukemias. nucleus to the cytoplasm, a mechanism first described by the group Leukemic stem cells (LSCs) seem to host biological properties that of Falini. LSC of NPM1mut patients might be the source for leukemic render them resistant to chemotherapy and thus might be self-renewal and might account to disease relapse after treatment. responsible for MRD.1 Because traditional chemotherapy often We were interested in the expression differences of the LSC- fails to eliminate LSC, development of novel therapeutics is enriched cell fraction descendent of NPM1mut and NPM1wt primary essential for the eradication of the complete leukemic cell fraction. AML patients. We screened our data with regard to new potential An immunotherapeutic approach could be one strategy to therapeutic structures to target LSC of NPM1mut patients. Our eliminate residual leukemic cells. focus was on targets for immunotherapeutic approaches that are Leukemia-associated antigens (LAAs) constitute targets for potential candidates for treatment of MRD. Functional immuno- immunotherapeutic approaches as they are involved in cell logical assays indicate the inhibition of CD34 þ CD38 À cells by differentiation and proliferation mechanisms.2 LAAs like RHAMM, immunotargeting strategies. proteinase 3 and Wilms’ tumor antigen 1 (WT1) were tested in We chose CD34 þ and CD38 À as surface markers for patients clinical peptide vaccination trials revealing immunological and and healthy volunteers to find access to the LSC-enriched cell clinical responses in patients with different hematological population. First, we screened our data to define expression malignancies.3 differences in the two AML groups NPM1mut and NPM1wt (Table 1). Recently, we described specific T-cell responses against Although most cells in NPM1mut AML are CD33 þ and CD34 À , the epitopes derived of the mutated region of nucleophosmin 1 CD34 þ CD38 À cell fraction harbors LSC within the NPM1mut (NPM1mut).4 Immune responses against the mutated protein of leukemic cells. Several groups demonstrated the presence of NPM1 might contribute to the favorable prognosis of acute NPM1mut in LSC and showed an adverse outcome in patients not myeloid leukemia (AML) patients carrying NPM1mut. We detected a carrying the NPM1mut confirming the prognostic value of mutated better overall survival in a smaller cohort of NPM1mut AML patients NPM1.9,10 Differentially expressed might influence showing immune responses against NPM1.5 We demonstrated molecularly and immunologically relevant pathways that could polyspecific cytotoxic T-cell (CTL) responses against several known have an impact on the better overall survival of the mutated LAAs but also NPM1#3 after preemptive donor lymphocyte patients. Therefore, we enriched the CD34 þ CD38 À cell infusion in an AML patient with NPM1mut and molecular population of 21 patient samples by fluorescence-activated relapse.6 cell sorting using 5 Â 107–2 Â 108 cells, depending on the cell Mutations in NPM1 are one of the most frequent molecular frequency (Figure 1a). CD34 þ CD38 À cell frequency in NPM1mut alterations in AML and therefore NPM1mut became an important AML patients (n ¼ 9) ranged from 0.63 to 6.8% (mean 3.87%)

Table 1. expression level analysis of the enriched LSC-containing fraction of NPM1mut, NPM1wt AML patients and HSC of healthy volunteers

Parametric LSC LSC HSC Fold- Fold-change Symbol Function P-value NPM1mut NPM1wt change HSC patients NPM1mut

0.0074418 249.32 25.71 14.6 9.7 17.05 SERPINA1 A serine protease inhibitor whose targets include elastase, plasmin, thrombin, trypsin, chymotrypsin and plasminogen activator 0.00005 205.93 30.08 27.7 6.85 7.43 H19 Encodes for noncoding RNA, may act as a tumor suppressor 0.0003405 92.67 14.89 — 6.22 — IGSF10 Cell adhesion, protein amino-acid phosphorylation 0.0016458 299.88 64.33 33.2 4.66 9.03 OSCAR Member of the leukocyte receptor complex protein family that has critical roles in the regulation of innate and adaptive immune responses 0.0035087 178.44 44.36 28.87 4.02 6.1 SUCNR1 Hematopoietic progenitor cell development 0.0005275 214.17 55.49 34.38 3.86 6.22 CD96 Member of the immunoglobulin superfamily; has a role in adhesive interactions of activated T and NK cells; functions in antigen presentation 0.0028565 146.39 56.39 — 2.6 — SH2D1A Has a role in the bidirectional stimulation of T and B cells 0.000834 70.45 37.78 24.28 1.87 2.9 IL12RB1 Lack associated with immunodeficiency Abbreviations: AML, acute myeloid leukemia; HSC, hematopoietic stem cell; LSC, leukemic stem cell; NPM1, nucleophosmin 1. Analysis of differences of both patient groups compared with the healthy HSC using BRB-ArrayTools, version 4.2.1 (Bethesda, MD, USA). Description of the gene function mentioned in the table refers to: http://www.ncbi.nlm.nih.gov/gene. Expression profiles of the two AML patient groups were compared with the expression levels in the HSC segment. We found several differentially expressed genes that are of immunological relevance. CD96 and IL12RB1 seem to be interesting targets for an immunotherapeutic approach, as they are overexpressed in enriched LSC fractions of NPM1mut AML patients.

Accepted article preview online 28 March 2014; advance online publication, 25 April 2014

& 2014 Macmillan Publishers Limited Leukemia (2014) 1736 – 1764 Letters to the Editor 1760

Patient A Patient B negative control

mut OCI AML 2 NPM1wt OCI AML 3 NPM1 all cells CD34+ cells

*** ***

**

**

**

p=0.0005

*

* * * *

* * * * *

Figure 1. (a) CD34/CD38 cell sorting of NPM1mut and NPM1wt. Frequency of CD34 þ CD38 À cells in NPM1mut patients before sorting is lower than that in NPM1wt patients. There is a trend in the difference of frequency (P ¼ 0.07). (b) Immunocytochemistry staining of the enriched LSC fraction for cytoplasmic NPM1. Enriched CD34 þ CD38 À cells of two representative AML patients (denominated A/B) with NPM1mut show cytoplasmic nucleophosmin stained in brownish color. The control samples (HV) shows no staining for NPM1 in the cytoplasm, but as expected NPM1 is detectable in the nucleus. (c) IL-12 influence on immunogenetic primed effector cells. Analyses of the immune-stimulating effect of IL-12 on activated CTL and their lyses potential on target cells. Cell lines OCI AML 2 (NPM1wt) and OCI AML 3 (NPM1mut) were used as target cells. There is no effect on adding IL-12 to target cells only. Lysis rate increases when effector cells are added. IL-12 seems to have an influence on the lysis potential of CTLs, the biggest effect was seen after 48 h (OCI2 P ¼ 0.0017; OCI3 P ¼ 0.0001). No significant difference could be seen between the two cell lines. (d) Flow cytometric CD96 expression analysis of 18 patients. The graph shows the mean CD96 expression of either all cells or CD34 þ cells only. Left: NPM1mut patients show a mean expression of CD96 on the cell surface of all cells of 24.35% compared with a mean expression of 7.2% in NPM1wt. Right: The mean expression of CD96 on the cell surface of CD34 þ cells of NPM1mut patients is 54% compared with 4.4% in NPM1wt patients. Expression differences in CD34 þ cells are highly significant (P ¼ 0.0005) comparing NPM1mut with NPM1wt patients. (e) CFU blockage by anti-CD96. CFU assays were performed using two AML patient samples with high blast counts. Addition of anti-CD96 antibody to blasts and effectors was inhibiting the growth of CFU comparable to the growth of blasts and effectors only (Pp0.05). Lysis through effector cells was most effective using 0.5 mg anti-CD96 and 0.05 mg human anti-mouse antibody (HAMA) (Pp0.05) with a mean difference of 82% in colony number. Anti-CD96 antibody or HAMA only had no effect on the colony formation (*P ¼ 0.05, **P ¼ 0.01, ***Pp0.001).

before sorting, after sorting purity increased to 91–97.6% (mean CD34 þ CD38 À cells in NPM1mut AML samples harbor cytoplasmic 93%). In NPM1wt patients (n ¼ 12), CD34 þ CD38 À frequency NPM1 demonstrated via immunocytochemical staining ranged between 0.78 and 86.2% (mean 33.08%) before sorting (Figure 1b), which indicates that these cells belong to the and after sorting from 91.3 to 98.4% (mean 95.2%). Enriched leukemic clone. We examined the expression of potentially

Leukemia (2014) 1736 – 1764 & 2014 Macmillan Publishers Limited Letters to the Editor 1761 relevant genes in LSC-enriched CD34 þ CD38 À cell fractions of LSC marker. IL-12 and anti-CD96 are also potential candidates NPM1mut compared with NPM1wt AML primary patients (Table 1). combining immunotherapeutic approaches—for example, with Interestingly, the differences are accentuated on genes that are vaccination strategies targeting NPM1, RHAMM, WT1 or Proteinase involved in T-cell immunity. Stronger immune responses in 3 or with other newly discovered T-cell-activating antibodies NPM1mut AML patients against leukemic cells and especially the mentioned above. The microarray analysis comparing enriched LSC fraction could be a factor for the better overall survival of LSC populations of NPM1wt and NPM1mut AML patients underlines NPM1mut patients compared with NPM1wt patients. Some of the the importance of the immune system in NPM1mut AML patients as detected genes are candidates for immunotherapeutic purposes, highly immunological regulatory pathways are differentially and we further analyzed their relevance in CD34 þ CD38 À cells regulated in NPM1mut compared with NPM1wt patients: For targeted by functional assays. example the NO2-dependent IL-12 pathway in natural killer cells We found several differentially expressed genes which are of (P ¼ 0.00077) and the Th1/Th2 differentiation pathway immunological relevance like immunoglobulin superfamily member (P ¼ 0.00133; data not shown). 10 (P ¼ 0.00034), CD96 (P ¼ 0.00052) and IL-12 receptor beta 1 Further, interesting genes without T-cell-mediated immuno- (IL12RB1, P ¼ 0.000834; Table 1). CD96 and IL12RB1 might logical relevance show differential expression in our microarray be interesting targets for immunotherapeutic approaches as analysis when NPM1mut AML patients were compared with they are overexpressed in enriched LSC fractions of NPM1mut NPM1wt. Several HOX genes, which are involved in the stem cell AML patients and therefore we examined them in further phenotype maintenance (data not shown),9,15 and other functional assays. interesting genes like SERPINA1 (P ¼ 0.006), OSCAR The IL12RB1 gene is an interesting target in the context of (P ¼ 6.11E À 05) and H19 (P ¼ 5.10E À 05) are all upregulated in enriched LSCs. The CD34 þ CD38 À cell fraction shows significantly the NPM1mut group (Table 1). higher IL12RB1 expression in AML with NPM1mut than with NPM1wt In summary, we could demonstrate that leukemic progenitor (1.87-fold; P ¼ 0.000834). Interleukin-12 (IL-12) is thought to have a cells of NPM1mut and NPM1wt AML patients can be effectively potent antitumor activity through immunostimulatory and anti- separated for array analysis. The difference in frequency of angiogenic mechanisms and/or direct activity on tumor cells. CD34 þ CD38 À -enriched LSC population in NPM1mut AML patients Furthermore, IL-12 is a proinflammatory and prostimulatory shows a trend (P ¼ 0.07). Through microarray analysis, we cytokine, which modulates the activity of many T-cell populations.11 detected differences in the expression of a relevant number of It has a fundamental effect on the direction of immune responses genes in leukemic progenitor cells of NPM1mut AML patients and variety of diseases by controlling the development of many compared with NPM1wt AML patients and also in normal HSC. contrasting T-cell populations. IL-12 also promotes generation of Markedly, genes with immunological functions seem to have an 11 mut proinflammatory T-helper type 1 and TH17 cells. IL12RB1 is important role in the NPM1 AML subtype. Besides peptides overexpressed in the LSC-enriched population of patients carrying derived from NPM1mut, which were already described as an the mutation, which might stimulate blasts, in the context of an immunogenic target for specific immunotherapy, further interest- immunogenic disease like NPM1mut AML, this effect might even be ing targets have been detected, like the cell surface marker CD96 enforced. The immune-stimulating effect of IL-12 was analyzed on and IL12RB1. Our functional assays suggest that these detected activated CTL and their lyses potential on target cells (Figure 1c). targets might have a therapeutic potential that has to be Cell lines OCI AML2 (NPM1wt)andOCIAML3(NPM1mut) were used as elucidated in clinical trials. In NPM1mut AML the important role target cells. The lysis rate is higher in the effector/target mix and the of the immune system becomes more and more evident. lyses effect is enforced by adding IL-12, showing the biggest effect after 48 h (OCI2 P ¼ 0.0017; OCI3 P ¼ 0.0001; Figure 1c). Further- more, IL-12 might directly inhibit the leukemic cell growth12 and CONFLICT OF INTEREST therefore the IL-12/IL12RB1 interaction might be of remarkable The authors declare no conflict of interest. relevance for leukemic cell rejection. A further candidate for LSC targeting is the cell surface marker CD96, a receptor that is specifically expressed on T cells in normal ACKNOWLEDGEMENTS hematopoiesis and is involved in T-cell activation. It is highly This work was supported by grants from the German Research Foundation (DFG upregulated upon T-cell activation and is expressed at low levels GR2676/3-1) and the Else Kro¨ner-Fresenius Stiftung (2011-A63). on resting T cells.13 In our analysis, CD96 showed a 4.26-fold higher expression in the NPM1mut compared with the NPM1wt CD34 þ CD38 À -enriched cell fraction (P ¼ 0.00052). We confirmed AUTHOR CONTRIBUTIONS our microarray findings by testing all patient samples via flow VS, LZ, JG and SH designed the study and wrote the manuscript, VS, LZ, JG, cytometry analysis using CD96 and CD34 staining (Supplementary LB and TB analyzed the data, VS, LZ, MR, MG, AL and UB performed the Figure S1). CD96 surface expression was 54% on CD34 þ cells in experiments, MW, KD, HS and HD provided advice on the study. the NPM1mut group and 4.4% in the wild-type group (P ¼ 0.0005; Figure 1d). We analyzed the effect of CD96 blockage by adding an V Schneider1, L Zhang1,2, L Bullinger1, M Rojewski3, S Hofmann1, anti-CD96 antibody to blasts and effectors; growth inhibition of M Wiesneth3, H Schrezenmeier3,MGo¨tz1, U Botzenhardt1, colony-forming unit (CFU) was comparable to blasts and effectors TFE Barth4,KDo¨hner1,HDo¨hner1 and J Greiner1,5 only (Pp0.05). The addition of CD96 and serum containing human 1Department of Internal Medicine III, University of Ulm, anti-mouse antibody enforced the growth reduction of CFU at Ulm, Germany; high concentration to a mean of 82% (Pp0.05; Figure 1e). CD96 2Department of Oncology, Tongji Hospital, Tongji Medical College, could be an interesting structure to target NPM1mut CD34 þ CD38 À Huazhong University of Science and Technology, Wuhan, China; cells as it is not relevantly expressed on hematopoietic stem cell 3Institute of Clinical Transfusion Medicine and Immunogenetics Ulm, (HSC) (Table 1). Recently, patients with hematological malignan- German Red Cross Blood Transfusion Service Baden-Wu¨rttemberg–Hessen cies and several solid tumors treated with T-cell-stimulating and Institute of Transfusion Medicine, University of Ulm, antibodies like Nivolumab14 and bispecific T-cell activity Ulm, Germany; antibodies like Blinatumomab showed encouraging and even 4Department of Pathology, University of Ulm, Ulm, Germany and enthusiastic antileukemic effects. CD96 could possibly be used as 5Department of Internal Medicine, Diakonie Hospital Stuttgart, a structure for targeted therapy, for example, using for a bispecific Stuttgart, Germany antibody. Clinical trials have to elucidate the clinical role of this E-mail: [email protected]

& 2014 Macmillan Publishers Limited Leukemia (2014) 1736 – 1764 Letters to the Editor 1762 REFERENCES 8 Schlenk RF, Dohner K, Krauter J, Frohling S, Corbacioglu A, Bullinger L et al. 1 Ossenkoppele G, Schuurhuis GJ. MRD in AML: time for redefinition of CR? Blood Mutations and treatment outcome in cytogenetically normal acute myeloid 2013; 121: 2166–2168. leukemia. N Engl J Med 2008; 358: 1909–1918. 2 Greiner J, Bullinger L, Guinn BA, Dohner H, Schmitt M. Leukemia-associated 9 Martelli MP, Pettirossi V, Thiede C, Bonifacio E, Mezzasoma F, Cecchini D et al. antigens are critical for the proliferation of acute myeloid leukemia cells. Clin CD34 þ cells from AML with mutated NPM1 harbor cytoplasmic mutated Cancer Res 2008; 14: 7161–7166. nucleophosmin and generate leukemia in immunocompromised mice. Blood 3 Anguille S, Van Tendeloo VF, Berneman ZN. Leukemia-associated antigens and 2010; 116: 3907–3922. their relevance to the immunotherapy of acute myeloid leukemia. Leukemia 2012; 10 Gerber JM, Smith BD, Ngwang B, Zhang H, Vala MS, Morsberger L et al. A clinically 26: 2186–2196. relevant population of leukemic CD34( þ )CD38(-) cells in acute myeloid leukemia. 4 Greiner J, Ono Y, Hofmann S, Schmitt A, Mehring E, Gotz M et al. Mutated regions Blood 2012; 119: 3571–3577. of nucleophosmin 1 elicit both CD4( þ ) and CD8( þ ) T-cell responses in patients 11 Vignali DA, Kuchroo VK. IL-12 family cytokines: immunological playmakers. with acute myeloid leukemia. Blood 2012; 120: 1282–1289. Nat Immunol 2012; 13: 722–728. 5 Greiner JSV, Schmitt M, Go¨ tz M, Do¨hner K, Wiesneth M, Do¨hner H et al. Immune 12 Ferretti E, Di Carlo E, Cocco C, Ribatti D, Sorrentino C, Ognio E et al. Direct responses against the mutated region of cytoplasmatic NPM1 might contribute to inhibition of human acute myeloid leukemia cell growth by IL-12. Immunol Lett the favorable clinical outcome of AML patients with NPM1 mutations (NPM1mut). 2010; 133: 99–105. Blood 2013; 122: 1087–1088. 13 Majeti R. Monoclonal antibody therapy directed against human acute myeloid 6 Hofmann S, Gotz M, Schneider V, Guillaume P, Bunjes D, Dohner H et al. leukemia stem cells. Oncogene 2011; 30: 1009–1019. Donor lymphocyte infusion induces polyspecific CD8( þ ) T-cell responses with 14 Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF et al. concurrent molecular remission in acute myeloid leukemia with NPM1 mutation. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J J Clin Oncol 2013; 31: e44–e47. Med 2012; 366: 2443–2454. 7 Cancer Genome Atlas Research Network. Genomic and epigenomic landscapes of 15 Alharbi RA, Pettengell R, Pandha HS, Morgan R. The role of HOX genes in normal adult de novo acute myeloid leukemia. N Engl J Med 2013; 368: 2059–2074. hematopoiesis and acute leukemia. Leukemia 2013; 27: 1000–1008.

Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)

Altered expression of tumor suppressor PHF20 in myeloproliferative neoplasms

Leukemia (2014) 28, 1762–1764; doi:10.1038/leu.2014.117 an alternative mechanism was suggested by our above- mentioned observation regarding PHF20 genomic loss in PMF. Our main objective was to find out whether PHF20 is downregulated in PMF and related MPN. Myeloproliferative neoplasms (MPN) arise from somatic mutations The current study was approved by the Mayo Clinic institutional at the level of the hematopoietic stem cell, which result in abnormal review board. For PHF20 mutation analysis, we used granulocyte expansion of the myeloid compartment. Clonal myelopoiesis in DNA (primers available upon request). For PHF20 expression MPN is accompanied by genomic instability, which promotes the analysis, peripheral blood mononuclear RNA was extracted accumulation of biologically more aggressive subclones that using the RNeasy Plus Mini Kit (Qiagen). Single-strand comple- ultimately undergo leukemic transformation. The molecular or mentary DNA was prepared using the High-Capacity cDNA biological mechanisms behind clonal evolution in MPN and other Reverse Transcription Kit (Life Technologies, Grand Island, NY, myeloid malignancies are complex and cumulative.1 USA). Custom primer express FAM/BHQ primer probe pair specific We recently reported the identification of several novel DNA for PHF20 and glyceraldehyde 3-phosphate dehydrogenase alterations in primary myelofibrosis (PMF), a subtype of MPN, (GAPDH) was obtained from Integrated DNA Technologies, Inc. using high-resolution mate-pair sequencing (HRMPS).2 In one (Coralville, IA, USA). Each reaction was performed in triplicate patient with del(20q), HRMPS revealed breakpoints at 20q11.22 at and each reaction included 7.5 ml of Taqman 2 Â MM position 34383499 and 20q13.2 at position 49891766. One of (Life Technologies), 1.5 ml of primer express probe/primer mix, these breakpoints involved the gene plant homeodomain finger 1 ml of cDNA and 5 ml of water. Reaction conditions were as 20 (PHF20), resulting in monoallelic genomic loss distal to exon 1. follows: 95 1C for 10 min, followed by 40 cycles of: 95 1C for 15 s PHF20 has recently been shown to be important in binding to and 60 1C for 1 min. We averaged the triplicate Ct values from and regulating the stability of p53.3–5 p53 is a master tumor each sample and normalized this to the average GAPDH Ct. We suppressor well known for its role in maintaining cellular measured the relative change by plotting 2 À DCt. Results were homeostasis.6 Loss of p53 leads to genomic instability, which analyzed using Prism6 GraphPad Software, Inc. (La Jolla, CA, USA). promotes tumorigenesis. p53 function is downregulated during We first screened the coding regions of PHF20 in seven cases of transformation of PMF into acute myeloid leukemia (AML) and PMF and did not find any evidence of mutations. We next studied decreased p53 levels in AML have been correlated with poor PHF20 expression level in 44 patients with BCR-ABL1-negative prognosis.7 Up to 15–20% of patients with post-PMF AML were MPN, including 24 with PMF (median age 62 years, range 40–79; reported to harbor somatic p53 mutations.8 However, p53 79% males), 11 with polycythemia vera (PV; median age 57 years, mutations are not frequent enough in hematological range 20–88; 46% males) and 9 with essential thrombocythemia malignancies (o15% overall) to account for the main (ET; median age 52 years, range 29–82; 33% males). We mechanism behind loss of p53.9 Similarly, the methylation status subsequently added four patients with post-PV MF (median age of cell cycle regulators may not be responsible for the 69 years, range 37–76; 25% males) and seven with myelodysplas- downregulation of p53 that is observed in PMF.10 Consequently, tic syndrome (MDS; median age 81 years, range 66–86; 100%

Accepted article preview online 28 March 2014; advance online publication, 25 April 2014

Leukemia (2014) 1736 – 1764 & 2014 Macmillan Publishers Limited