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. 2015 Oct 12;28(4):486-499.
doi: 10.1016/j.ccell.2015.09.001. Epub 2015 Sep 24.

hnRNP K Is a Haploinsufficient Tumor Suppressor that Regulates Proliferation and Differentiation Programs in Hematologic Malignancies

Miguel Gallardo  1 , Hun Ju Lee  2 , Xiaorui Zhang  1 , Carlos Bueso-Ramos  3 , Laura R Pageon  4 , Mark McArthur  4 , Asha Multani  5 , Aziz Nazha  1 , Taghi Manshouri  1 , Jan Parker-Thornburg  5 , Inmaculada Rapado  6 , Alfonso Quintas-Cardama  1 , Steven M Kornblau  1 , Joaquin Martinez-Lopez  6 , Sean M Post  7
Affiliations

hnRNP K Is a Haploinsufficient Tumor Suppressor that Regulates Proliferation and Differentiation Programs in Hematologic Malignancies

Miguel Gallardo et al. Cancer Cell. .

Abstract

hnRNP K regulates cellular programs, and changes in its expression and mutational status have been implicated in neoplastic malignancies. To directly examine its role in tumorigenesis, we generated a mouse model harboring an Hnrnpk knockout allele (Hnrnpk(+/-)). Hnrnpk haploinsufficiency resulted in reduced survival, increased tumor formation, genomic instability, and the development of transplantable hematopoietic neoplasms with myeloproliferation. Reduced hnRNP K expression attenuated p21 activation, downregulated C/EBP levels, and activated STAT3 signaling. Additionally, analysis of samples from primary acute myeloid leukemia patients harboring a partial deletion of chromosome 9 revealed a significant decrease in HNRNPK expression. Together, these data implicate hnRNP K in the development of hematological disorders and suggest hnRNP K acts as a tumor suppressor.

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Conflict of interest statement

Conflicts of interests: The authors declare no conflicts.

Figures

Figure 1
Figure 1. HNRNPK expression is reduced in patients with AML that harbor a 9q deletion
Quantitative RT-PCR analysis of HNRNPK levels from bone marrow aspirates of AML patients who carry 9q deletions (n = 12) and healthy donors controls (n = 8) (p = 0.0001). Data are represented as mean ± SEM determined from triplicate samples after normalization to geometric mean of β-ACTIN and GAPDH. ***p < 0.005. P-values were calculated using the Mann-Whitney test.
Figure 2
Figure 2. Generation of Hnrnpk haploinsufficient mice
(A) Schema of the Hnrnpk locus and the KOMP targeting vector (Hnrnpk1(KOMP)Wtsi). The targeting vector replaced exons 3 through 6 and with a neomycin selectable marker flanked by LoxP sites (ovals) and LacZ cassette flanked by Flp sites (arrowheads). Black numbered boxes denote Hnrnpk exons. (B) PCR analysis of targeted recombination in Hnrnpk+/− mice. The 5.9-kb amplicon (dark blue arrow heads) represents the recombined Hnrnpk allele using external and internal primers for the 5′ arm. Wild-type DNA serves as a negative control. The 10.1-kb (black arrow heads), 9.1-kb (red arrow heads), 8.1-kb (green arrow heads), and 5.5-kb (light blue arrow heads) amplicons represent the recombined Hnrnpk allele using external and internal primers for the 3′ arm. See also: Figure S1.
Figure 3
Figure 3. Hnrnpk+/− mice have developmental defects and reduced hnRNP K expression
(A) Observed and expected number of Hnrnpk+/− and wild type mice at weaning (21 days). (B) Growth retardation in Hnrnpk+/− mice at day 3 (Left panel) and 10 days (Right panel). (C) Weight (in grams) of Hnrnpk+/− (n = 10) and wild type littermates (n = 8) over the first four weeks of life (p = 0.0001). (D) Weight analysis (in grams) of adult Hnrnpk+/− and wild type mice stratified by sex (Female, p = 0.011) and (Male, p = 0.003). (E) Observed and expected ratio of Hnrnpk−/−, Hnrnpk+/−, and wild type mice from 12 separate hnRNP K+/− matings at weaning (21 days). (F) Quantitative RT-PCR analysis of Hnrnpk expression in the bone marrow of wild type (n = 4) and Hnrnpk+/− (n = 4) mice (p = 0.0396). (G) Western blot analyses of hnRNP K levels in lysates from wild type and Hnrnpk+/− mice, (upper-left panel: MEFs; upper-right panel: whole bone marrow; bottom-left panel: spleen; bottom-right panel: liver). Data are represented as mean ± SEM. *p < 0.05 and ***p < 0.005. p-values were calculated using Mann-Whitney test.
Figure 4
Figure 4. Reduced hnRNP K expression results in increased proliferation and a dampened p21 response following DNA damage
(A) Increased cell proliferation in Hnrnpk+/− MEFs. Three low passage (P2) MEF cell lines per genotype were plated for 24, 48 or 72 hr, and then assayed by WST-8 (p = 0.0070). Each data point represents the mean ± SEM for three separate MEF lines per genotype (B) Quantitative RT-PCR analysis of p21 levels before and after irradiation from WT and Hnrnpk+/− MEFs. (C) Western blot analysis of hnRNP K and p21 expression in lysates from γ-IR treated wild type and Hnrnpk+/− MEFs. β-actin serves as an internal control. Data are represented as mean ± SEM. ***p < 0.005. P-values were calculated using unpaired t-tests and Mann-Whitney tests. See also, Figure S2.
Figure 5
Figure 5. Hnrnpk+/− mice develop myeloid hyperplasia
(A) Cell blood count (CBC) of peripheral blood comparing neutrophils (p = 0.0006) and basophils (p = 0.0477) from wild type (n = 8) and Hnrnpk+/− (n = 11) mice and platelets (p = 0.0095) of wild type (n = 6) and hnRNP K+/− (n = 6) mice. (B) Flow cytometry analysis of double positive Ly6G/CD11b myeloid populations (50,000 gated cells) in the peripheral blood of wild type (n = 6) and hnRNP K+/− (n =6) (upper panel, p = 0.0354) and bone marrow of wild type (n = 7) and hnRNP K+/− (n = 13) (bottom panel, p = 0.0008) mice. Percentages were compared and analyzed using Mann-Whitney test. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.005. See also, Figure S3.
Figure 6
Figure 6. Hnrnpk+/− mice have reduced survival and are tumor prone
(A) Kaplan-Meier curves indicating survival of Hnrnpk+/− (n = 76) and wild type (n = 37) mice. Statistical significance was determined by log rank test (p = 0.0001). (B) Hnrnpk haploinsufficiency results in tumor phenotypes (n = 55; 62% myeloproliferation, 31% lymphoma and 4% hepatocellular carcinoma). (C) Hematoxylin and Eosin (H&E) staining of paraffin-embedded bone marrow sections and peripheral blood (PB) smears from wild type Hnrnpk+/− mice diagnosed with myeloid hyperplasias. The scale bar for H&E staining represents 50 μm and 25 μm for PB smears. (D) H&E staining of paraffin-embedded splenic sections and PB smears from wild type and lymphoma burdened Hnrnpk+/− mice. (E) H&E and immunohistochemical staining of malignant T-cell (CD3 positive, Bottom Panel) and B-cell (B220 positive, Top Panel) lymphomas in the liver of Hnrnpk+/− mice. (F) H&E staining of paraffin-embedded liver sections from wild type and Hnrnpk+/− mice diagnosed with hepatocellular carcinoma. See also Figure S4.
Figure 7
Figure 7. hnRNP K-mediated activation of cytokines contributes to proliferation and differentiation phenotypes
(A) Cytokines array analysis of IL-3, IL-6, G-CSF and GM-CSF levels in the peripheral blood of wild type (n = 10) and Hnrnpk+/− mice (n = 9) diagnosed with myeloid hyperplasia (IL-3: p = 0.0002, IL-6: p = 0.0004, G-CSF: p = 0.0002, and GM-CSF: p = 0.0002). (B) Analyses of colony formation assays after 14 days using Lin-CD117+ hematopoietic stem cells from wild type (n = 8) and Hnrnpk+/− (n = 17) mice (number of colonies: p = 0.0078 and number of cells: p = 0.0001). All experiments were performed in triplicate. Data are represented as mean ± SEM. **p < 0.01 and ***p < 0.005. P-values were calculated using Mann-Whitney test. (C) Replating analysis of Lin-CD117+ hematopoietic stem cells from wild type (n = 5) and Hnrnpk+/− (n = 7) mice. Each passage was analyzed every 14 days. All experiments were performed in quadruplicate. Data are represented as mean ± SEM. (D) Flow cytometry analysis of CD45.1+ (host) and CD45.2+ (donor) in the transplanted host mice and B220+ and Ly6G+ cells within the CD45.2+ population. (E) CBC analysis of untransplanted NSG (control) mice and NSG mice transplanted with wild type or Hnrnpk+/− HSCs. (F) Wright's staining of peripheral blood (PB) smears from NSG mice transplanted with wild type or Hnrnpk+/− HSCs harboring malignant lymphoid cells. See also, Figure S5.
Figure 8
Figure 8. Hnrnpk haploinsufficiency alters expression of genes controlling proliferation and differentiation
(A) Western blot analysis of C/EBPα and C/EBPβ from lysates of wild type and Hnrnpk+/− mice. Upper arrow denotes the p42 isoform of C/EBPα and the lower arrow marks the p30 isoform. β-actin serves as a loading control. (B) Quantitative RT-PCR analysis of C/EBPα and C/EBPβ levels from whole bone marrows of wild type (n = 8) and Hnrnpk+/− (n = 10) mice (C/EBPα: p = 0.042 and C/EBPβ: p = 0.020). (C) Quantitative RT-PCR analysis of p21 levels in wild type (n = 8) and Hnrnpk+/− (n = 10) bone marrows (p = 0.0044). (D) Immunohistochemical analysis of phos-Y705Stat3 levels in the bone marrow wild type and Hnrnpk+/− mice diagnosed with myeloid hyperplasia. The scale bar represents 50 μm. (E) ChIP analysis of hnRNP K interacting with the C/EBPα, C/EBPβ and p21 genes in wild type or Hnrnpk+/− tissues. Each rectangle represents the corresponding gene. The closed black box denoted the region harboring a putative hnRNP K binding sequence that is amplified by the corresponding primer sets. The “check-mark” denotes positive hnRNP K binding and the “X” denotes a lack of interaction. (F) Quantitative RT-PCR analysis of TNFα levels in spleens from wild type (n = 7) and tumor burdened Hnrnpk+/− (n = 5) mice (p = 0.0177). (G) H&E staining and immunohistochemical analyses of B220, TNFα, and phos-Y705STAT3 expression in Hnrnpk+/− mice with malignant lymphoid infiltration in the liver. Data are represented as mean ± SEM. *p < 0.05 and ***p < 0.005. P-values were calculated using unpaired t and Mann-Whitney tests. See also Figure S6.
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