The absent/low expression of CD34 in NPM1-mutated AML is not related to cytoplasmic dislocation of NPM1 mutant protein

INTRODUCTION NPM1-mutated acute myeloid leukemia (AML) represents about one-third of all adult AML [1] and, due its unique clinicopathological and genetic features [2], is recognized as a leukemia entity of the World Health Organization (WHO) Classification of myeloid neoplasms. Nucleophosmin (NPM1) is a multifunctional protein physiologically located in the nucleolus [2]. NPM1 mutations, the most common genetic lesion in AML, abrogate the ability of the protein to localize in the nucleolus and create a new nuclear export signal (NES) at the C-terminus, leading to enhanced nuclear export of mutant NPM1 and its aberrant accumulation in the cytoplasm of leukemic cells [1, 3, 4]. We demonstrated that the interaction between mutant NPM1 and the nuclear exporter Exportin-1 (XPO1) causes the aberrant cytoplasmic delocalization of mutant NPM1 and is responsible for the high expression of HOX genes in NPM1-mutated AML, since relocalization of the NPM1 mutant by XPO1 inhibitors causes early downregulation of HOX genes that is followed by cell differentiation and growth arrest [2, 5]. The rapid loss of HOX expression, despite XPO1 inhibition does not restore the physiologic localization of NPM1 to the nucleolus [5], strongly suggest that the interaction between XPO1 and mutant NPM1 (rather than its localization) is responsible for maintaining high HOX levels. Another characteristic feature of NPM1-mutated AML is the absent/low expression of CD34 [6, 7] that yet remains poorly investigated. This feature, combined with the low HLA-DR [8], strong CD33 expression [9] and presence of abnormal PML bodies [10], is reminiscent of acute promyelocytic leukemia (APL) and have inspired APL-like treatment strategies (i.e., all-trans retinoic acid and arsenic trioxide) also in NPM1-mutated AML both preclinically [10] and in patients (NCT04689815, NCT03031249). Unlike HOX genes, CD34 expression seems to be independent from XPO1-mediated cytoplasmic dislocation of mutant NPM1. This is supported by the finding that in most NPM1-mutated AML patients, a small subset of early CD34+ hematopoietic precursors carrying NPM1 mutations/cytoplasmic NPM1 is usually present [11], suggesting a derivation from CD34+ hemopoietic stem cells, with the potential of multilineage differentiation. On the other hand, the observation that at least a percentage of NPM1-mutated AML may derive from CD34-negative hematopoietic stem cells, raises the question of a possible relationship between absent/low expression of CD34 and cytoplasmic dislocation of mutant NPM1 [12]. Clinically, CD34 expression in NPM1-mutated AML has been mostly associated with an adverse outcome [13]. To address this issue, we performed functional studies to assess whether the nuclear relocalization of the mutant NPM1 could result in the re-expression of CD34. Moreover, we searched for CD34+/NPM1 cytoplasmic precursors in the bone marrow (BM) biopsies of NPM1-AML patients at diagnosis and relapse, using a highly specific monoclonal antibody against mutant NPM1. The results of these studies are presented below.

in mycoplasma-free conditions and maintained in 5% CO2 at 37°C at a concentration of 0.5x10 6 cells/ml. Xenograft (PDX) NPM1-mutated AML cells derived from patients after the signature of a written informed consent form, were grown in IMDM medium supplemented with 10% FBS, 1% Glu, 1% P/S and cytokines (30ng/ml FLT3L, 30ng/ml G-SCF and 15ng/ml TPO (all Cell Guidance Systems)) and maintained at a concentration of 2.0x10 6 cells/ml.

Treatment of OCI-AML3 and PDX cells with Selinexor
OCI-AML3 and PDX NPM1-mutated AML cells were plated at 0.5x10 6 cells/ml and treated with 50nM Selinexor or 0.3% DMSO (control) for 24h. At 12 and 24 hours, 0.2x10 6 cells were collected for flow cytometry analyses. 0.5x10 6 cells were collected in RNA lysis buffer for total RNA purification and subsequent analyses. 0.1x10 6 cells were spotted onto poly-Llysine coated glass slides for subsequent immunofluorescence analysis.

Flow cytometry
In each experiment, 0.2x10 6 cells were washed with PBS and stained for 20min at 4°C in the dark with the PE-conjugated anti-CD34 antibody (BD Biosciences) to assess CD34 expression levels. Excess antibody was removed by washing the cells with PBS. In all samples, cell debris were excluded based on FSC-A/SSC-A dot-plot and 7-Amino-Actinomycin D (7-AAD, BD Biosciences) was used for dead-cell exclusion. Singlets were gated using the FSC-A/FSC-H dot-plot. Flow cytometry was performed with the BD FACSCanto II. Dynamics of NPM1c degradation upon dTAG-13 treatment was determined by GFP expression analysis. Flow cytometry data were analyzed with FlowJo 10.7 software (BD Biosciences) and expressed as fold change of the median fluorescence intensity (MFI) of the treated sample, relative to the control one. Graphs generation and statistical analyses were performed using Prism 8 (GraphPad).

Cell sorting of patient' cells
Leukemic cells were obtained from peripheral blood and isolated after written informed consent (Perugia Hospital) from a 32-year-old female patient with newly diagnosed NPM1mutated AML. FITC-conjugated anti-CD34 and PerCP/Cy5.5-conjugated anti-CD45 antibodies (Beckman Coulter) were used for flow cytometry analysis and cell sorting. CD34+ and CD34-cells were sorted using the BD FACS Aria III cell sorter. Purity was higher than 90% in both fractions. Sorted cells were spotted onto poly-L-lysine coated glass slides and used for subsequent immunofluorescence analysis.

RNA isolation and qRT-PCR gene expression analysis
Total RNA was isolated according to manufacturer's instructions using the Quick-RNA Microprep Kit (Zymo Research) including in-column DNase I treatment. RNA quality control and quantification was performed using the NanoDrop spectrophotometer. RNA was reverse transcribed with the SuperScript IV First-Strand Synthesis System Kit (Invitrogen).

Immunoblot analysis
Cell pellets were lysed with 2X Laemmli sample buffer (Bio-Rad) and boiled at 95°C for 5min. Total cell lysates and 5μl of the Precision Plus Protein Dual Color Standards (Bio-Rad) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a 4-15% Mini-PROTEAN TGX Precast Protein Gel (Bio-Rad) and transferred onto PVDF membranes using the Trans-Blot Turbo transfer system (Bio-Rad). After 1h blocking in 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T), membranes were probed overnight at +4°C with the following primary antibodies diluted in 5% non-fat dry milk: antiβ-tubulin (monoclonal mouse, 1:1000, T4026 Merck); anti-tNPM1 (rabbit polyclonal, 1:1000, HPA011384 Merck). Following primary antibody incubation, membranes were washed and probed with anti-mouse or anti-rabbit Horseradish Peroxidase (HRP)-conjugated secondary antibodies, were imaged using the Luminata Crescendo Western HRP Substrate (Merck Millipore) system and were visualized on a ChemiDoc MP imaging system (Bio-Rad).
Images were captured on the chemiluminescence setting and analyzed using the Image Lab software.   Immunoblot analysis of dTAG OCI-AML3 cells treated with DMSO (control) or dTAG-13 500nM for 12 to 72 hours. Anti-tNPM1 antibody, recognizing either wild type NPM1 and mutant NPM1 fused to FKBP F36V and GFP, was used to confirm the selective degradation of mutant NPM1 in the dTAG-13 treated cells. β-Tubulin was used as loading control. E.

Bone marrow samples and immunohistochemistry (IHC) analysis
Immunoblot analysis of dTAG IMS-M2 cells treated with DMSO (control) or dTAG-13 500nM for 12 to 72 hours. Anti-tNPM1 antibody, recognizing either wild type NPM1 and mutant NPM1 fused to FKBP F36V and GFP, was used to confirm the selective degradation of mutant NPM1 in the dTAG-13 treated cells. β-Tubulin was used as loading control.