Recurrent cyclin D2 mutations in myeloid neoplasms

V Khanna^{1

2

3}, C A Eide^{1

4

5}, C E Tognon^{1

4

5}, J E Maxson^{1

4

6}, B Wilmot^{1

7}, D Bottomly^{1

7}, S McWeeney^{1

7}, D K Edwards V^{1

4

6}, B J Druker^{1

4

5}, J W Tyner^{1

4

6}

Affiliations

¹ Knight Cancer Institute, Oregon Health and Science University, Portland, OR, USA.
² Howard Hughes Medical Research Fellows Program, Howard Hughes Medical Institute, Portland, OR, USA.
³ Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, USA.
⁴ Division of Hematology and Medical Oncology, Oregon Health and Science University, Portland, OR, USA.
⁵ Howard Hughes Medical Institute, Portland, OR, USA.
⁶ Department of Cell, Developmental and Cancer Biology, Oregon Health and Science University, Portland, OR, USA.
⁷ Division of Bioinformatics and Computational Biology, Department of Medical Informatics and Clinical Epidemiology, Oregon Health and Science University, Portland, OR, USA.

PMID: 28630439
PMCID: PMC5763909
DOI: 10.1038/leu.2017.195

Recurrent cyclin D2 mutations in myeloid neoplasms

V Khanna et al. Leukemia. 2017 Sep.

. 2017 Sep;31(9):2005-2008.

doi: 10.1038/leu.2017.195. Epub 2017 Jun 20.

Authors

Affiliations

¹ Knight Cancer Institute, Oregon Health and Science University, Portland, OR, USA.
² Howard Hughes Medical Research Fellows Program, Howard Hughes Medical Institute, Portland, OR, USA.
³ Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, USA.
⁴ Division of Hematology and Medical Oncology, Oregon Health and Science University, Portland, OR, USA.
⁵ Howard Hughes Medical Institute, Portland, OR, USA.
⁶ Department of Cell, Developmental and Cancer Biology, Oregon Health and Science University, Portland, OR, USA.
⁷ Division of Bioinformatics and Computational Biology, Department of Medical Informatics and Clinical Epidemiology, Oregon Health and Science University, Portland, OR, USA.

PMID: 28630439
PMCID: PMC5763909
DOI: 10.1038/leu.2017.195

No abstract available

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figures

**Figure 1**
CCND2 P281 variants are resistant to degradation and nuclear export. (a) Protein extracts from NIH-3T3 cells stably expressing each construct (top labels) were treated with cycloheximide (CHX) and were analyzed by western blotting using an antibody against CCND2. The wild-type CCND2 construct shows clear protein reduction by 60 min post-CHX treatment, while mutant constructs show relatively stable levels of protein, even by 120 min post-CHX treatment. (b) Representative immunofluorescence images of NIH-3T3 cells stably expressing either wild-type or CCND2 P281 variants. Cells were serum starved for 20 h in RPMI containing 0.1% FBS, reintroduced to serum, and then collected for immunofluorescence analysis at the specified time points post-serum reintroduction. Eight hours and 16 h post-reintroduction were chosen as collect time points because they represent time points at which the majority of wild-type CCND2 is nuclear and cytoplasmic, respectively. Red =CCND2, blue =DAPI. (c) Quantification of mean pixel density in the nucleus to establish nuclear localization of CCND2 constructs. At least ten cells were analyzed for each condition. ***P<0.0001 by one-way ANOVA and subsequent Tukey’s post hoc tests. Error bars show s.d.

**Figure 2**
Assessment of the oncogenicity and CDK4/6 inhibitor sensitivity of CCND2 P281 variants. (a) IL-3 withdrawal assay demonstrating the dependence of all CCND2-expressing lines on IL-3 for proliferation. (b) Normalized cell viability (relative to day 0 of IL-3 withdrawal) over time. Cells expressing CCND2 constructs show delayed decreases in viability relative to cells expressing an empty control vector. (c) Representative images of wells containing murine hematopoietic colonies grown in Methocult M3534 (supplemented with murine IL-3, IL-6 and SCF). Images were taken after 14 days of incubation. (d) Quantification of colony number, performed in triplicate, for murine hematopoietic cells expressing the labeled constructs. Colony number was compared between groups via one-way ANOVA followed by Tukey’s post hoc tests. **P<0.01 for comparison of colony number in empty vector-expressing cells versus the number in P281L-expressing cells. *P<0.05 for comparison of colony number in empty vector-expressing cells versus the number in P281L-expressing cells. No significant differences in colony numbers existed between wild-type CCND2-expressing cells and P281 variant-expressing cells. Error bars show s.d. (e) Sensitivity of CCND2-expressing NIH-3T3 cells to CDK4/6 inhibition. Cells expressing cyclin D2 constructs show increased sensitivity to palbociclib (a CDK4/6 inhibitor) relative to parental NIH-3T3 cells. Error bars show standard error of the mean for each data point.

See this image and copyright information in PMC

References

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Recurrent cyclin D2 mutations in myeloid neoplasms

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