Novel NPM1 exon 5 mutations and gene fusions leading to aberrant cytoplasmic nucleophosmin in AML

The nucleophosmin (NPM1) gene, encoding for a multifunctional shuttling protein located in the nucleolus,¹ is mutated in approximately one-third of acute myeloid leukemias (AMLs).^2,NPM1 mutations affect the last exon (exon 12) almost exclusively and cause changes at the C-terminus end (loss of tryptophans and creation of a nuclear export signal [NES] motif) leading to the aberrant dislocation of mutant NPM1 (and wild-type [WT] NPM1) in the cytoplasm of AML cells (NPM1c⁺).^3-5 This event is easily detectable by immunohistochemistry (IHC) in paraffin-embedded biopsies.^6,7 Rare NPM1 mutations involving exons 9⁸ and 11,⁹ as well as the NPM1/MLF1 fusion generated by t(3;5)(q25;q34)¹⁰ and NPM1/HAUS1 fusion created by t(5;18)(q35;q21),¹¹ also lead to cytoplasmic NPM1, indicating that this event is critical for leukemogenesis.^12,13 We report novel NPM1 mutations and fusion transcripts in AML and discuss their functional significance and the best approach to identify them.

From 2005 to 2019, 929 unselected AML patient samples afferring to Perugia (PG patients) from different Italian centers were studied by IHC in paraffin-embedded bone marrow (BM) biopsies, western blot (WB) with anti-NPM1 antibodies (supplemental Figure 1), and NPM1 exon 12 Sanger sequencing^6,14 (screening A). On IHC in BM biopsies, 387 (41.6%) of 929 samples were NPM1c⁺. NPM1c⁺ cases that were exon 12 WT and/or negative by WB with antibodies against NPM1 exon 12 mutants¹⁴ were regarded as discrepant and further investigated. We also screened an additional 141 patients with AML from the Munich Leukemia Laboratory (MLL) who were selected based on the following criteria: (1) normal/intermediate karyotype, (2) no NPM1 exon 12 mutations, and (3) absence of genetic alterations mutually exclusive with NPM1 mutations.¹⁵ All 141 cases were investigated by targeted sequencing for NPM1 exons 1 to 12 by a next-generation sequencing (NGS) TruSeq custom panel (Illumina; screening B).

Finally, we characterized 3 new NPM1 rearrangements found within 13 979 AML patient samples analyzed by chromosome banding analysis in routine diagnostics, followed by fluorescence in situ hybridization and RNA sequencing¹⁶ (screening C). BM biopsies were not available from patients included in screening B and C cohorts, because this examination is not routinely performed in some centers. The study was conducted in accordance with the Declaration of Helsinki and received local institutional review board approval.

The newly identified NPM1 mutants and fusion transcripts were cloned into pEGFP-C1 vectors and further studied functionally by ectopic expression in murine fibroblast NIH-3T3 cells.^3,12 Details are given in the supplemental Methods.

Screening A identified a diagnostic discrepancy in 5 (1.3%) of 387 NPM1c⁺ AML cases (Figure 1A-B; data not shown). Sanger sequencing and/or targeted sequencing of the NPM1 coding sequence detected an exon 11 mutation in 1 patient (data not shown),^9,17 involvement of exon 9⁸ in another patient (supplemental Figure 2), and 3 novel mutations involving exon 5 in the other 3 cases (PG patients 1, 2, and 3; Figure 1C). Specifically, PG patient 1 showed a 21-nucleotide in-frame insertion at c408-409 (F,5'-GCGGAGGATGTGAAACTCTTA) producing a new mutant NPM1 protein, 7 aa longer than WT (p.L136_137insAEDVKLL), whereas PG patient 2 displayed an 18-nucleotide out-of-frame insertion at c409-410 (F,5'-AATGATCTGTCACTTCTG), with the creation of a new stop codon leading to a truncated protein of 137 aa (p.S137_K137fs*). In PG patient 3, the exon 5 mutation consisted of a 27-nucleotide in-frame insertion at c424-425 (F,5'-TTTCTGCCTTAAGTATATCTGGAAAGC), with the production of a new NPM1 mutant protein 9 aa longer than WT (p.K141_142insLSALSISGK; Figure 1C).

Screening B identified a fourth exon 5 mutation in another patient (MLL patient 4; Figure 1C). In this case, 2 in-frame insertions/duplications, each consisting of 9 nucleotides, occurred at c399-400 (F,5'-CAACTCTTA) and c400-401 (F,5'-GTGGGCTGC), leading to a new NPM1 mutant protein 6 aa longer than WT (p.K134_Q134insLLSGLQ; Figure 1C).

Notably, with the exception of PG patient 2, all other patients with NPM1 mutants retained the C-terminus of NPM1 WT, including the NoLS (Figure 1A-C; supplemental Figures 3 and 4). This is a unique finding, because all NPM1 mutant cases reported so far were found to carry a disrupted NoLS resulting from loss of both tryptophans 288 and 290 or 288 alone.^3,12 Despite retaining NoLS, these exon 5 mutants showed cytoplasmic localization both by IHC (Figure 1A) and when transfected as GFP-NPM1 mutants in NIH-3T3 (supplemental Figures 3 and 4). Cytoplasmic dislocation was promoted by an extra NES with a high score¹⁸ (super NES) that was able to counteract the NoLS driving the mutant to the nucleolus (Figure 1C). This was also confirmed by the pRev(1.4)-eGFP NES efficiency assay, as previously described¹² (supplemental Figure 5). NES-dependent cytoplasmic localization was proven by relocation into the nucleus/nucleoli upon exportin-1 inhibition by leptomycin B (supplemental Figure 6). In addition to cytoplasmic localization, all new mutant samples except for PG patient 2 also retained a nucleolar localization both in transfected cells (Figure 1C; supplemental Figures 3E and 4E) and IHC (PG patients 1 and 3; Figure 1A), likely to have resulted from the intact NoLS.

In screening C, 3 new NPM1 translocations were identified: t(5;10)(q35;q23), t(5;18)(q34;q12), and t(5;6)(q35;q23), generating the NPM1/RPP30 (MLL patient 1), NPM1/SETBP1 (MLL patient 2), and NPM1/CCDC28A (MLL patient 3) fusion transcripts, respectively (Figure 2A-D). NPM1/CCDC28A was also retrospectively detected in 1 of the Italian patients, who at screening A showed a diagnostic discrepancy for NPM1 between IHC and molecular analysis (PG patient 4; Figure 2C-D; supplemental Figure 7). This fusion was recently reported but not further characterized in a pediatric patient,¹⁹ confirming its recurrence in AML. The encoded fusions were predicted to be out of frame for NPM1/RPP30 but in frame for both NPM1/SETBP1 and NPM1/CCDC28A (Figure 2A-B; supplemental Figure 8). The NPM1/CCDC28A fusion protein was shown to be expressed by WB in MLL patient 1 and located in the cytoplasm of AML cells by IHC in PG patient 4 (Figure 2C). In all cases, NPM1 was involved as an upstream gene so that, in the derived protein, the original C-terminus of NPM1 containing the NoLS was lost (Figure 2A-B; supplemental Figure 8). Strikingly, with the exception of MLL patient 2 (carrying NPM1/SETBP1, with previous RUNX1/RUNX1T1 AML, and in complete molecular remission at time of analysis), a C-terminal NES, either newly created (in NPM1/RPP30) or present in the partner protein (in NPM1/CCDC28A), was added to the original NPM1 protein sequence (Figure 2A-B; supplemental Figures 8 and 9). Accordingly, the corresponding GFP-NPM fusion proteins (supplemental Figure 10) showed an NES-dependent cytoplasmic localization only for NPM1/RPP30 and NPM1/CCDC28A (Figure 2; supplemental Figure 6). The NPM1/SETBP1 transcript in MLL patient 2 indeed spontaneously disappeared within 1.2 years, calling into doubt its pathogenetic role in AML.

In conclusion, we report novel NPM1 mutations and fusion transcripts in AML and demonstrate that, similarly to exon 12 mutations, they act to delocalize NPM1 in the cytoplasm, further supporting our original hypothesis that this event is critical for leukemogenesis.^4,12,13 Other biological similarities with AML mutated at NPM1 exon 12 include de novo occurrence, CD34 negativity, morphological pattern (multilineage dysplasia,²⁰ increased megakaryocytes²¹), and type of associated mutations²² (Figures 1D and 2D).

Our findings also raise the question of how to recognize these rare variants.^17,23 As diagnostic workflow for AML at presentation, we recommend that, in addition to standard molecular analyses according to European LeukemiaNet²⁴ 2017, a BM biopsy is also performed. If IHC with a monoclonal antibody against the NPM1 N-terminus clearly shows cytoplasmic positivity for NPM1 but no NPM1 exon 12 mutations are detected by conventional molecular assays, all other NPM1 exons should be sequenced by NGS. If no rare NPM1 mutations are detected, fluorescence in situ hybridization/RNA sequencing will allow identification of NPM1-containing fusion transcripts. For centers not performing BM biopsy or IHC at diagnosis, the only way to detect these rare NPM1 mutations is to perform targeted NGS in all AMLs, by including in the analysis NPM1 exons other than 12. This should be feasible, because NGS, although recommended by European LeukemiaNet 2017 only in the context of clinical trials, is now increasingly used, especially in Western countries.

Because of their rarity (∼1%), the clinical impact of these novel NPM1 genetic alterations remains unclear, and its assessment will require specific analysis in large clinical trials. Moreover, identification of these very rare NPM1 mutations/rearrangements would allow minimal residual disease monitoring by designing patient-specific quantitative reverse transcription polymerase chain reaction assays.¹⁷ 

The authors thank Claudia Tibidò and Laura Pinacoli for secretarial assistance.

This work was supported by Associazione Italiana per la Ricerca sul Cancro 2016 Investigator Grant 18568 (B.F.), European Research Council (ERC) 2016 advanced grant 740230 (B.F.), and ERC 2016 Consolidator Grant 725725 (M.P.M.). This article is dedicated to the memory of Roberta Pacini.

Contribution: M.P.M. and B.F. conceived the study; R.R., A.V., V.M.P., G.M., R.C., E.V., L.B., C.Q., F. Mezzasoma, I.G., and F. Milano performed the experiments and analyzed the data; O.S., V.C., F.A., G.S., C.V., F.D.R., O.A., G.A., A.R., and F.F. provided patient samples; G.M. and S.A. carried out histological analyses, with technical support from R.P., A.T., and B.B.; E.T. and P.S. supervised molecular analyses; M.M., T.H., and C.H. identified the new translocations and provided samples; M.P.M. and B.F. wrote the manuscript; and all authors contributed to writing and approval of the manuscript.

Conflict-of-interest disclosure: B.F. licensed a patent on NPM1 mutants (102004901256449). B.F. and M.P.M. declare honoraria from Rasna Therapeutics, Inc., for scientific advisor activities. M.P.M. also declares honoraria/consultancy and scientific advisory board activities for AbbVie, Amgen, Celgene, Janssen, Novartis, Pfizer, and Jazz Pharmaceuticals. V.M.P. declares consultancy and scientific advisory board activities for Novartis. L.B. and C.V. declare consultancy and scientific advisory board activities for AbbVie. F.D.R. declares honoraria/consultancy and scientific advisory board activities for Jazz Pharmaceuticals, Bristol-Myers Squibb, Janssen, and Novartis. O.A. declares honoraria/consultancy and scientific advisory board activities for Janssen, Novartis, Celgene, and Amgen. A.R. declares honoraria/consultancy and scientific advisory board activities for Novartis, Amgen, Pfizer, Gilead, Celgene/Bristol-Myers Squibb, Sanofi, Jazz Pharmaceuticals, TEVA, Italfarmaco, Omeros, and Coimmune. P.S. declares honoraria/consultancy and scientific advisory board activities for AbbVie, Janssen, Novartis, AstraZeneca, and Incyte. T.H., C.H., and M.M. declare part ownership of Munich Leukemia Laboratory. The remaining authors declare no competing financial interests.

Correspondence: Brunangelo Falini, Centro di Ricerche Emato-Oncologiche (CREO), University of Perugia, Ospedale S Maria della Misericordia, S Andrea delle Fratte 06132 Perugia, Italy; e-mail: brunangelo.falini@unipg.it; and Maria Paola Martelli, Centro di Ricerche Emato-Oncologiche (CREO), University of Perugia, Ospedale S Maria della Misericordia, S Andrea delle Fratte 06132 Perugia, Italy; e-mail: maria.martelli@unipg.it.