ALBERT

Description: Acute myeloid leukemia expressing mutated NPM1 gene and cytoplasmic nucleophosmin (NPMc+ AML) [Falini B et al, NEJM2005;352:254–266] is a new entity of WHO classification that shows distinctive biological and clinical features, including a unique molecular signature characterized by downregulation of CD34 and upregulation of most HOX genes [Falini B et al, Blood2007;109:874–885]. Involvement of HOX genes in the maintenance of the stem-cell phenotype strongly suggest that AML with mutated NPM1 originates from a multipotent hematopoietic progenitor (HSC). This view is also supported by immunohistological findings showing that AML with mutated NPM1 frequently displays multilineage involvement [Pasqualucci L et al, Blood2006;108:4146–4155]. On the other hand, the frequent negativity of NPMc+ AML for the HSC-associated antigen CD34 raises the question of whether the mutation event occurs in a CD34-negative HSC (these cells have been identified in mice) or whether a minimal pool of CD34-positive NPM1-mutated leukemic cells does exist. Currently, the hierarchical level of stem cell involvement in NPMc+ AML is unknown. To address this issue, we purified CD34+ cells from NPMc+ AML patients and detected NPM1 mutant protein in the sorted population by Western blot with anti-NPM mutant specific antibodies [Martelli MP et al, Leukemia 2008] (Figure 1A). We investigated 6 NPMc+ AML patients presenting at diagnosis with 0.12%, 0.14%, 0.38%, 5%, 22%, and 28% of CD34+ cells in the peripheral blood. In all cases, CD34+ fractions (purity 〉90%) harboured NPM1 mutant protein, indicating they belong to the leukemic clone (Figure 1B). The percentage of most undifferentiated CD34+/CD38− cells in the CD34+ fractions ranged from 5 to 97%. Notably, in at least one case, all CD34+ NPM1-mutated leukemic cells were CD38−negative. Moreover in all cases, CD34+ NPM1-mutated leukemic cells appeared to express CD123 (IL-3 receptor), considered a marker of the leukemic stem cell and target of potential therapy. Double staining of bone marrow biopsies with anti-CD34 and anti-NPM antibodies revealed that the rare CD34+ cells expressed NPM1 aberrantly in the cytoplasm. Inoculation of CD34+ NPM1-mutated AML cells into sublethally irradiated NOD/SCID mice resulted into leukemia engrafment in various body sites, especially bone marrow, spleen, lung and liver. Preliminary results showed that CD34+ leukemic cells reacquired the same leukemic phenotype as the original patient’s, including CD34-negativity of the leukemic bulk in spite of any lack of differentiation. This finding suggests that NPM1 mutant protein may be involved in downregulation of CD34 antigen, while keeping a gene expression profile typical of the hematopoietic stem cell. These findings suggest the CD34+ fraction contains the SCID-leukemia initiating cells (SL-IC) and point to CD34+/CD38− HSC as the cell of origin of AML with mutated NPM1. Figure Figure

Description: Acute myeloid leukemia (AML) with mutated NPM1 shows distinctive biologic and clinical features, including absent/low CD34 expression, the significance of which remains unclear. Therefore, we analyzed CD34+ cells from 41 NPM1-mutated AML. At flow cytometry, 31 of 41 samples contained less than 10% cells showing low intensity CD34 positivity and variable expression of CD38. Mutational analysis and/or Western blotting of purified CD34+ cells from 17 patients revealed NPM1-mutated gene and/or protein in all. Immunohistochemistry of trephine bone marrow biopsies and/or flow cytometry proved CD34+ leukemia cells from NPM1-mutated AML had aberrant nucleophosmin expression in cytoplasm. NPM1-mutated gene and/or protein was also confirmed in a CD34+ subfraction exhibiting the phenotype (CD34+/CD38−/CD123+/CD33+/CD90−) of leukemic stem cells. When transplanted into immunocompromised mice, CD34+ cells generated a leukemia recapitulating, both morphologically and immunohistochemically (aberrant cytoplasmic nucleophosmin, CD34 negativity), the original patient's disease. These results indicate that the CD34+ fraction in NPM1-mutated AML belongs to the leukemic clone and contains NPM1-mutated cells exhibiting properties typical of leukemia-initiating cells. CD34− cells from few cases (2/15) also showed significant leukemia-initiating cell potential in immunocompromised mice. This study provides further evidence that NPM1 mutation is a founder genetic lesion and has potential implications for the cell-of-origin and targeted therapy of NPM1-mutated AML.

Description: Key Points The NPM1 mutant affects megakaryocytic development in mice. NPMc+ mutant mice mimic some features of human NPM1-mutated AML.

Description: Key Points ATRA and ATO affect NPM1 protein levels in AML cells and induce cell growth inhibition and apoptosis. AML cells with mutated NPM1 respond to ATRA/ATO, and this might be exploited therapeutically.

Description: Abstract 4135 Acute myeloid leukemia (AML) expressing mutated NPM1 gene and cytoplasmic nucleophosmin (NPMc+ AML) [Falini B et al, NEJM 2005;352:254-266] is a new entity of WHO classification that shows distinctive biological and clinical features. AML with mutated NPM1 usually presents with a high white blood cell count; the bone marrow biopsy is usually markedly hypercellular and leukemic cells frequently show myelomonocytic or monocytic features, with dysplasia and involvement of two or more cell lineages in about 25% of cases. Lack, or low expression, of CD34 in over 90% of cases is the most distinctive immunophenotypic feature of NPM1-mutated AML and is independent of leukemic cell maturation. NPM1 gene mutation without concomitant FLT3-ITD identify a subgroup of AML patients with a favorable prognosis and has been associated with an approximately 50-60% probability of survival at 5 years in younger patients. Here we report 4 out of 41 (10%) patients, admitted at our Hospital in the last year, with new-diagnosed AML with mutated NPM1 presenting with life-threatening thromboembolic (either arterial or venous) events. The main characteristics of these patients are summarized in Table 1. The patients had neither personal nor family history concerning thromboembolism. Hyperleukocytosis was a common feature of the vast majority of NPM1-mutated AML patients at diagnosis. Immunophenotypic analysis did not show a peculiar phenotype in these patients. Table 1 Characteristics of patients with NPM1-mutated AML and thrombosis. Case report no Age Sex (M/F) FAB subtype WBC/mmc Type of thrombosis Site of thrombosis 1 41 F M1 14970 arterial Anterior interventricular branch of left coronary artery 2 56 M M4 93990 arterial external iliac and femoral (right limb) 3 63 M M2 113000 deep venous great saphenous veins (bilateral) 4 73 F M4 190000 deep venous iliac and femoral In two patients (cases 1 and 2), the arterial thromboembolic event (acute myocardial infarction and acute ischemia of right lower limb, respectively) presented about one month before diagnosis of leukemia. In the other 2 patients (cases 3 and 4), deep venous thromboembolism was concomitant with the diagnosis of leukemia. One patient (case 4), who could not initiate chemotherapy for severe concomitant renal failure, died few days after diagnosis. The other patients recovered from the acute event and upon diagnosis of leukemia were promptly treated with standard polychemotherapy which allowed to obtain complete hematological remission associated with complete resolution of the thromboembolic event. The clinical course after chemotherapeutic treatment of the patients outlines the importance and life saving role of early chemotherapy even under adverse circumstances. The pathogenesis of thromboembolic disease in hematological malignancies is complex and multifactorial: tumor cell-derived procoagulant, fibrinolytic or proteolytic factors and inflammatory cytokines affect clotting activation. Other important factors include infectious complications and hyperleukocytosis. However, large vessel thrombosis is a very rare clinical presentation. Our report of severe thromboembolic events at presentation in AML with mutated NPM1 suggests some still unidentified biological features of this leukemia which we are currently investigating. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2621 Acute myeloid leukemia (AML) carrying nucleophosmin (NPM1) gene mutations account for about one-third of adult AML, show distinctive biological and clinical features and have been included as a provisional entity in the 2008 World Health Organization (WHO) classification of myeloid neoplasms. In spite of the relatively good prognosis of NPM1-mutated AML, there are still cases that show poorer outcome, especially those associated with FLT3-ITD mutation and elderly patient population. Therefore new therapeutic strategies need to be explored. As for other types of cancer, evidence is emerging that, besides the genomic DNA alterations, epigenetic dysregulation may play a role in AML pathogenesis. We investigated the effects of sodium butyrate, a short-chain fatty acid which has long been known to be a histone deacetylase inhibitor (HDACi) able to induce maturation in normal and tumor cells, in cellular models of NPM1-mutated AML in vitro: i) the OCI/AML3 cell line, previously identified as a human AML cell line carrying cytoplasmic mutated NPM1 in the absence of FLT3-ITD; ii) primary AML cells originated from a patient with NPM1-mutated AML bearing FLT3-ITD mutation (MONT1) and propagated as cell line in NOD/SCID mice; and iii) primary AML cells from NPM1-mutated AML patients at diagnosis. In either cell lines or patients' primary AML cells carrying NPM1 mutation, but not in the U937 or OCI/AML2 cell lines (not harboring NPM1 gene mutation) used as control, growth arrest, cell cycle arrest (G0-G1 phase) and pro-apoptotic effects were evident after 24 hrs and marked after 48 hrs of treatment with doses of drug of 0.5–1 mM. No signs of differentiation were evident at morphological and flow cytometric examinations of treated cells. Western blot analysis with specific antibodies showed that levels of either NPM1 mutant or wild-type protein did not appear significantly affected by treatment with sodium butyrate. Interestingly, induction of apoptosis was associated with marked activation of caspase-8, suggesting involvement of the death cell receptors pathway. Indeed, flow cytometric analysis showed 2-fold increased expression of TRAIL-receptor DR5 upon drug treatment at 48 hrs. Moreover, concomitant treatment with a specific caspase-8 inhibitor prevented sodium butyrate induced-cell growth arrest and markedly reduced apoptosis in OCI/AML3 cell line. Derivatives of butyrate, which are capable of acting as HDAC inhibitors, are currently being investigated in clinical trials; preliminary results suggest their potential applicability in cancer treatment. Here we investigated the effect of panobinostat (LBH589), a pan HDACi, in human AML cells lines and show results similar to those observed upon sodium butyrate treatment in OCI/AML3 cells. In particular, we show that panobinostat induces dose-dependent cell growth arrest, with G0-G1 block, associated with p21 protein induction, and apoptosis, associated with TRAIL-receptor DR5 upregulation and activation of caspase 8, in AML cells harboring NPM1 gene mutation. However, unlike what was observed with sodium butyrate, panobinostat induced also dowregulation of NPM1 mutant (and to a lesser extent, wild type) protein. Mechanisms underlying this phenomenon are under investigation. In particular, the possible role of acetylation of the heat shock protein Hsp90 (as a consequence of panobinostat-mediated HDAC6 inhibition) with disruption of its chaperone functions followed by instability of its client proteins, including cytoplasmic NPM1, is explored. Disclosures: Falini: Xenomics: Patents & Royalties.

Description: Abstract 480 Acute myeloid leukemia with mutated NPM1 gene and cytoplasmic nucleophosmin (NPMc+ AML) [Falini B et al, NEJM 2005;352:254-266] is a new entity of WHO classification that shows distinctive biological and clinical features [Falini B et al, Blood 2007;109:874-885] which include negativity for CD34 antigen expression at both immunohistochemistry and gene expression profiling. Flow cytometric analysis shows that, in most NPM1-mutated AML, percentages of CD34+ cells are in the low range (〈 5-10%). Detection of NPM1 mutations by molecular techniques and/or immunohistochemistry and Western Blot analysis with specific antibodies provides an important tool for tracking the genetic lesion in leukemic cells at different hierarchical stage. We previously reported involvement by NPM1 gene mutation of the CD34+ cell fraction isolated from patients with NPM1-mutated AML, and, in one case, the involvement, in particular, of the early progenitor CD34+/CD38- [Martelli MP et al, Blood (ASH Annual Meeting Abstracts) 2008;112:307]. Here we expand and confirm our previous observation in 5 cases of CD34-negative NPM1-mutated AML. CD34+/CD38- cells were isolated by either FACS (3 cases, purity 〉98%) or MACS-sorting (2 cases, purity 〉92%) and analyzed by molecular analysis and Western Blot with a specific anti-NPM1 mutant antibody, respectively. The presence of either NPM1 gene mutation or mutant protein was demonstrated in all samples analyzed proving the CD34+/CD38- cells belong to the leukemic clone. This cell subpopulation displayed also immunophenotypic features classically associated to leukemic stem cells (LSCs) (CD123+/CD33+/CD90-) in all (16/16) samples analyzed, suggesting they might actually represent the LSCs in NPM1-mutated AML. Indeed, CD34+ cell fraction isolated from NPM1-mutated AML was able to generate leukemia in immunocompromised mice resembling the original patient's disease. However, there is experimental evidence that, at least in some CD34-negative AML, also the CD34- population may contain LSCs. Whether the CD34- cell compartment in NPM1-mutated AML is also able to engraft and outgrow into leukemia in mice remains to be clarified. For this purpose, we assessed the engraftment ability of CD34- cells from 5 NPM1-mutated AML patients. No engraftment was observed in one case. Interestingly, in three patients with myelomonocytic (M4, 2 cases) and myelocytic (M2, 1 case) AML, the CD34- fraction resulted into marrow engraftment by human CD45+/CD33+ myeloid cells that, at morphological and immunohistological grounds, consisted of a mixed population of macrophage cells expressing the CD68 (PG-M1) antigen and mature looking myeloperoxidase (MPO)-positive cells. This pattern possibly reflects short-term engraftment by leukemic cells devoid of self-renewal potential that differentiated into mature elements. However, the neoplastic nature of engrafted cells could be established with certainty only in one case by western blotting detection of NPM1 mutant protein. Immunohistochemistry could not help in these cases to establish the leukemic nature of human cells since terminally differentiated leukemic cells in NPM1-mutated AML show nucleus-restricted NPM1 positivity. In contrast, the pure CD34+ fraction (availabel for comparison in one of these three cases) engrafted as AML with clear blastic morphology and cytoplasmic dislocation of nucleophosmin. In a fourth patient, the highly purified CD34- fraction from relapsed NPM1-mutated AML engrafted in mice with a typical AML picture. These preliminary findings suggest that in general the CD34- fraction from NPM1-mutated AML may have more limited engraftment potential than the CD34+ fraction. Further studies are ongoing to address this issue. Disclosures: Falini: Xenomics: Patents & Royalties.

Description: NPM1 mutations are among the most common mutations in acute myeloid leukemia (AML), being found in about 30% of de novo AML in adults. NPM1 is a multifunctional nucleolar chaperone involved in genomic stability and ribosome biogenesis. All the mutations in NPM1 described so far result in cytoplasmic protein localization (NPM1c) through the acquisition of a nuclear export signal (NES) at the C-terminus. Based on these observations, we hypothesized that cytoplasmic localization of NPM1c is necessary for AML pathogenesis and maintenance. To test our hypothesis, we edited the C-terminus of the NPM1 mutant allele in order to remove the NES and re-localize NPM1c to the nucleus. Specifically, using our recently optimized CRISPR-Cas9 approach (Gundry, et al. Cell Reports 2016), we sought to introduce indels to disrupt the C-terminal NES in AML cells bearing NPM1 mutation A (heterozygous 4bp insertion at the C-terminus), thereby creating novel edited alleles encoding for a mutant NPM1 with nuclear localization. Cell lines, primary derived xenografts (PDX) and primary AML samples harboring NPM1 mutation A were successfully edited with an sgRNA spanning the 4bp insertion. While the NPM1 wild-type allele remained intact, the mutant allele was edited with up to 90% efficiency. The novel edited alleles could direct nuclear localization of a GFP-NPM1 fusion construct. Additionally, re-localization of cytoplasmic NPM1 in edited cells was confirmed by immunofluorescence. Return of NPM1 protein to the nucleus resulted in terminal differentiation, and significantly impaired cell growth, colony forming ability and engraftment in xenograft models. Transcriptome analysis on two cell lines with mutated NPM1 revealed that upon nuclear re-localization of NPM1c, only 22 genes were down regulated more than 2 fold in both cell lines. Strikingly, 11 of these 22 genes were HOX genes. Altogether these data indicate that the cytoplasmic localization of NPM1c is necessary to maintain the transcriptional signature and leukemic phenotype of AML cells with mutated NPM1 . To extend our study further, we used homology-directed repair (HDR) to successfully correct NPM1 mutations to the wild type sequence in AML cell lines (Fig.1). We simultaneously fused GFP to the C-terminus of the protein to verify its appropriate localization (Fig.1). Moreover, to assess whether the phenotype of AML cells depends on the amount of mutant NPM1 in the cytoplasm, we inserted a series of NPM1 mutant alleles (a, b and c) that resulted in different proportions of cytoplasmic versus nuclear localization (i.e. a - no cytoplasmic mutant NPM1, b - ~50% cytoplasmic/50% nuclear mutant NPM1, c - ~100% cytoplasmic mutant NPM1; Fig.1) in two different AML cell lines with mutated NPM1 . Cells with high levels of nuclear NPM1 tended to differentiate rapidly, while cells with a greater proportion of cytoplasmic NPM1 remained less differentiated and continued to proliferate (Fig.1). This established an ongoing competition in which cells with higher nuclear NPM1 were rapidly outcompeted by unedited cells, while cells with higher cytoplasmic NPM1 were not. Thus, these experiments clearly demonstrate the dependence of the leukemic phenotype on the cytoplasmic localization of NPM1. Recently, compounds able to inhibit the nuclear exporter CRM1/XPO1 have been developed and are currently being tested in clinical trials (e.g. selinexor). Since NPM1 is shuttled to the cytoplasm by CRM1/XPO1, its inhibition re-localizes NPM1c to the nucleus. We therefore treated cell lines, PDX and primary samples with selinexor with the aim to reproduce the nuclear re-localization observed using CRISPR-Cas9. As expected, selinexor treatment resulted in clear growth arrest, differentiation, and NPM1c re-localization with dynamics similar to what was seen with gene editing. Importantly, after only 3 days of treatment selinexor produced a dramatic drop of HOXA, HOXB and MEIS1 expression. In conclusion, allele-specific editing is a powerful tool to probe the mechanistic aspects of oncogenic dependencies. By achieving nuclear re-localization of mutant NPM1, we demonstrated that cytoplasmic localization of NPM1c is necessary for NPM1-mutant AML cells to maintain their leukemic phenotype. Drugs promoting mutant NPM1 nuclear localization are therefore attractive candidates for clinical treatment of AML with mutated NPM1 . Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.