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Natural killer (NK) cells are innate lymphocytes with cytotoxic activity. Understanding the factors regulating cytotoxicity is crucial for improving NK-cell adoptive therapies. Here, we studied a previously unknown role of p35 (CDK5R1), a coactivator of cyclin-dependent kinase 5 (CDK5) in NK-cell function. p35 expression was thought to be neuronal-specific and the majority of studies are still focused on neuronal cells. Here, we show that CDK5 and p35 are expressed in NK cells and are kinase-active. NK cells from p35 knockout mice were analyzed and showed significantly increased cytotoxicity against murine cancer cells, while they did not show any differences in cell numbers or maturation stages. We confirmed this using human NK cells transduced with p35 short hairpin RNA (shRNA), showing similar increase in cytotoxicity against human cancer cells. Overexpression of p35 in NK cells resulted in moderate decrease in cytotoxicity, while expressing a kinase-dead mutant of CDK5 displayed increased cytotoxicity. Together, these data suggest that p35 negatively regulates NK-cell cytotoxicity. Surprisingly, we found that TGFβ, a known negative regulator of NK-cell cytotoxicity, induces p35 expression in NK cells. NK cells cultured with TGFβ exhibit reduced cytotoxicity, while NK cells transduced with p35 shRNA or mutant CDK5 expression exhibited partial reversal of this inhibitory effect pointing to an interesting hypothesis that p35 plays an important role in TGFβ-mediated NK-cell exhaustion. Significance: This study reports a role for p35 in NK-cell cytotoxicity and this might help to improve NK-cell adoptive therapy.

Overexpression of kinase-dead CDK5 increases NK92 cytotoxicity while overexpression of p35 decreases cytotoxicity. A, Western blot analysis of protein from NK92 cells overexpressing CDK5 (OE CDK5), kinase-dead mutant CDK5 (OE CDK5-K33T), p35 (OE p35), or empty vector control (OE Ctrl). B, NK92 cells were plated in complete media with a starting count of 250,000 cells. Live cell count was obtained after 48 hours cell culture via trypan blue staining and manual counting on a hemocytometer. ns, not significant. Graphs display mean ± SD, n = 3 biological cultures, one-way ANOVA with Dunnett multiple comparisons test. C, NK92 cells were stained with Annexin V and PI and analyzed via flow cytometry. Percentage of cells staining positive for Annexin V and/or PI were graphed. Graphs display mean ± SD, n = 3 biological cultures. D, NK92 cells were fixed in 70% ethanol and stained with PI, then analyzed via flow cytometry. G0–G1, S, and G2–M phases of the cell cycle are indicated. E, NK92 cells (OE Ctrl, OE CDK5, OE CDK5-K33T) were cocultured with fluorescently-labeled Jeko-1 cells for 16 hours at different E:T ratios, followed by staining with PI and gating on labeled cancer cells to measure cancer cell death via flow cytometry. ****, P < 0.0001. Graph displays mean ± SD, n = 3 biological cocultures, two-way ANOVA with Dunnett multiple comparisons test. F, NK92 cells (OE Ctrl, OE p35) were cocultured with fluorescently-labeled Jeko-1 cells for 16 hours at 5:1 E:T ratio, followed by staining with PI and gating on labeled cancer cells to measure cancer cell death via flow cytometry. *, P < 0.05. Graph displays mean ± SD, n = 3 biological cocultures, unpaired two-tailed t test.

Natural killer (NK) cells are innate lymphocytes with cytotoxic activity. Understanding the factors regulating cytotoxicity is crucial for improving NK-cell adoptive therapies. Here, we studied a previously unknown role of p35 (CDK5R1), a coactivator of cyclin-dependent kinase 5 (CDK5) in NK-cell function. p35 expression was thought to be neuronal-specific and the majority of studies are still focused on neuronal cells. Here, we show that CDK5 and p35 are expressed in NK cells and are kinase-active. NK cells from p35 knockout mice were analyzed and showed significantly increased cytotoxicity against murine cancer cells, while they did not show any differences in cell numbers or maturation stages. We confirmed this using human NK cells transduced with p35 short hairpin RNA (shRNA), showing similar increase in cytotoxicity against human cancer cells. Overexpression of p35 in NK cells resulted in moderate decrease in cytotoxicity, while expressing a kinase-dead mutant of CDK5 displayed increased cytotoxicity. Together, these data suggest that p35 negatively regulates NK-cell cytotoxicity. Surprisingly, we found that TGFβ, a known negative regulator of NK-cell cytotoxicity, induces p35 expression in NK cells. NK cells cultured with TGFβ exhibit reduced cytotoxicity, while NK cells transduced with p35 shRNA or mutant CDK5 expression exhibited partial reversal of this inhibitory effect pointing to an interesting hypothesis that p35 plays an important role in TGFβ-mediated NK-cell exhaustion.Significance:This study reports a role for p35 in NK-cell cytotoxicity and this might help to improve NK-cell adoptive therapy.

TGFβ induces p35 expression in a dose- and time-dependent manner. A, NK92 cells were incubated with fluorescently-labeled Jeko-1 cells and different concentrations of TGFβ for 16 hours. Cells were stained with PI and gated on labeled cancer cells via flow cytometry to measure cancer cell death. **, P < 0.01; ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cocultures, one-way ANOVA with Dunnett multiple comparisons test. B, NK92 cells were incubated with different concentrations of TGFβ in complete media for 48 hours. Protein was collected and analyzed via Western blot analysis. C, NK92 cells were incubated with 5 ng/mL TGFβ in complete media for 24 hours with or without the TGFβ receptor inhibitor EW-7197 (5 ng/mL). Protein was collected and analyzed via Western blot analysis. D, 1° NK cells were incubated in complete media with different concentrations of TGFβ and/or 5 ng/mL EW-7197 for 48 hours. Protein was collected and analyzed via Western blot analysis. E, 1° NK cells were cultured in cytokine-free media for 48 hours, followed by culture with either 200 U/mL IL2 or 5 ng/mL TGFβ. Protein lysate was collected at the indicated timepoints.

NK92 cells with p35 knockdown or CDK5-K33T overexpression mitigate TGFβ-induced inhibition of cytotoxicity. A, NK92 cells (shGFP or shp35) were cultured for 48 hours in the presence of 2.5 ng/mL TGFβ or PBS (−TGFβ). They were then cocultured with fluorescently-labeled Jeko-1 cells at 5:1 E:T ratio for 16 hours in the continued presence of 2.5 ng/mL TGFβ or PBS (−TGFβ). Cells were stained with PI and gated on labeled cells to measure cancer cell death. Average cytotoxicity percentage is overlaid on the corresponding bar. ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cocultures, multiple unpaired two-tailed t tests with correction for multiple comparisons using Holm-Šídák method. B, Supernatant from NK92 cell culture (shGFP or shp35) with or without Jeko cells was collected after 16 hours. Cytokine concentration was determined using a flow-based, multiplex human cytokine release assay. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Graphs display mean ± SD, n = 3 biological cocultures, multiple unpaired two-tailed t tests with correction for multiple comparisons using Holm-Šídák method. C, NK92 cells (OE ctrl, OE CDK5, OE CDK5-K33T, or OE p35) were cultured for 48 hours in the presence of 2.5 ng/mL TGFβ or PBS (−TGFβ). They were then cocultured with fluorescently-labeled Jeko-1 cells at 5:1 E:T ratio for 16 hours in the continued presence of 2.5 ng/mL TGFβ or PBS (−TGFβ). Cells were stained with PI and gated on labeled cells to measure cancer cell death. Average cytotoxicity percentage is overlaid on the corresponding bar. **, P < 0.01; ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cocultures, two-way ANOVA with Tukey multiple comparisons test. D, Supernatant from NK92 cell culture (OE ctrl, OE CDK5, OE CDK5-K33T, or OE p35) with or without Jeko cells was collected after 16 hours. Cytokine concentration was determined using a flow-based, multiplex human cytokine release assay. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cocultures, two-way ANOVA with Tukey multiple comparisons test.

Murine NK cells from p35 k/o mice display heightened cytotoxicity. A, Murine splenocytes or bone marrow cells from WT or p35 k/o mice were stained for CD3 and NK1.1 and mixed with counting beads, then analyzed via flow cytometry. Gating on NK1.1+CD3− cells, as well as the counting beads, allowed determination of mNK concentration and the total mNK cell count in each compartment. ns, not significant. Graphs display mean ± SD, n = 3 mice, unpaired two-tailed t test. B, mNK cells were isolated from WT, p35 k/o, or p35 Het mice and cocultured with calcein AM-labeled B16F10 cells at different E:T ratios for 4 hours. Fluorescence intensity of supernatant corresponding to calcein release from dead cells was measured to calculate NK-mediated cytotoxicity. **, P < 0.01. Graphs display mean ± SD, n = 3 biological cocultures (n = 2 for WT 1:1 ratio, p35 k/o 1:1 ratio, and p35 k/o in WT versus p35 Het versus p35 k/o experiment), and multiple unpaired two-tailed t tests with correction for multiple comparisons using Holm-Šídák method (WT vs. p35 k/o) or one-way ANOVA with Tukey multiple comparisons test (WT vs. p35 Het vs. p35 k/o). C, Murine splenocytes or bone marrow cells were stained for CD11b, CD27, CD3, and NK1.1 and analyzed via flow cytometry after gating on NK1.1+CD3− cells. Representative flow plots are shown. Percentages of mNK cells in each subset (CD11b+CD27−, CD11b−CD27+, and CD11b+CD27+) for each mouse were recorded and graphed. ns, not significant. Graphs display mean ± SD, n = 8 mice, multiple unpaired two-tailed t tests with correction for multiple comparisons using Holm-Šídák method. D, Murine splenocytes were stained for CD3, NK1.1, and NKG2D, NKp46, NKG2A, 2B4, LY6C, or LY49C/F/I/H. Representative histograms of activating/inhibitory receptor expression are shown, gated on live NK1.1+CD3− cells. E, Isolated mNK cells pooled from multiple mice were seeded at a concentration of 2e6 cells/mL and cultured ex vivo in RPMI media supplemented with 1,000 U/mL IL2. An additional 1,000 U/mL IL2 was added 2 days later without changing the media, followed by collection of supernatant after culture for an additional 2 days. Cytokine concentration was determined using a flow-based, multiplex murine cytokine release assay. ns, not significant; *, P < 0.05; **, P < 0.01. Graphs display mean ± SD, n = 3 biological cultures, unpaired two-tailed t tests.

CDK5 knockdown inhibits NK92 survival while p35 knockdown increases NK92 cytotoxicity. A, qRT-PCR results using RNA isolated from NK92 cells transduced with p35 shRNA (shp35) or CDK5 shRNA (shCdk5) compared with shGFP control. GAPDH was used as the housekeeping gene. Graphs display mean ± SD, n = 3 technical replicates. B, Western blot analysis of protein from NK92 cells transduced with p35 shRNA (shp35) or negative control GFP shRNA (shGFP). C, Phase contrast microscopy of NK92 cells transduced with CDK5 shRNA or one of two p35 shRNA clones, compared with shGFP control. Images are 10x magnification; scale bar, 100 μm. D, NK92 cells were plated in complete media with a starting count of 250,000 cells. Live cell count was obtained after 48 hours cell culture via trypan blue staining and manual counting on a hemocytometer. ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cultures, one-way ANOVA with Dunnett multiple comparisons test. E, NK92 cells were stained with Annexin V and PI and analyzed via flow cytometry. Percentage of cells staining positive for Annexin V and/or PI were graphed. ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cultures, one-way ANOVA with Dunnett multiple comparisons test. F, NK92 cells were cocultured with fluorescently-labeled Jeko-1 cells for 16 hours to measure the effect of p35 knockdown on cytotoxicity. Cells were stained with PI and gated on the labeled cancer cells to measure cancer cell death. ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cocultures, two-way ANOVA with Dunnett multiple comparisons test.

Supplementary Methods, Figures 1-4, Table 1 from Simultaneous Visualization of Protumorigenic Src and MT1-MMP Activities with Fluorescence Resonance Energy Transfer

Supplementary Figure 1 from Membrane Type-1 Matrix Metalloproteinase Confers Aneuploidy and Tumorigenicity on Mammary Epithelial Cells

Supplementary Movie 3 from Simultaneous Visualization of Protumorigenic Src and MT1-MMP Activities with Fluorescence Resonance Energy Transfer

Supplementary Table 1 from Membrane Type-1 Matrix Metalloproteinase Confers Aneuploidy and Tumorigenicity on Mammary Epithelial Cells

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Supplemental Figures. This file contains supplemental figures concerning analysis of megakaryocyte progenitor and erythroid progenitor analysis in the leukemia marrow, platelet and megakaryocyte progenitor analysis after DBZ treatment of leukemia mice, as well as CXCL12 promoter CHIP analysis

Supplemental Figure Legends

More than half of T-cell acute lymphoblastic leukemia (T-ALL) patients harbor gain-of-function mutations in the intracellular domain of Notch1. Diffuse infiltration of the bone marrow commonly occurs in T-ALL and relapsed B-cell acute lymphoblastic leukemia patients, and is associated with worse prognosis. However, the mechanism of leukemia outgrowth in the marrow and the resulting biologic impact on hematopoiesis are poorly understood. Here, we investigated targetable cellular and molecular abnormalities in leukemia marrow stroma responsible for the suppression of normal hematopoiesis using a T-ALL mouse model and human T-ALL xenografts. We found that actively proliferating leukemia cells inhibited normal hematopoietic stem and progenitor cell (HSPC) proliferation and homing to the perivascular region. In addition, leukemia development was accompanied by the suppression of the endosteum-lining osteoblast population. We further demonstrated that aberrant Notch activation in the stroma plays an important role in negatively regulating the expression of CXLC12 on osteoblasts and their differentiation. Notch blockade reversed attenuated HSPC cycling, leukemia-associated abnormal blood lineage distribution, and thrombocytopenia as well as recovered osteoblast and HSPC abundance and improved the hematopoietic-supportive functions of osteoblasts. Finally, we confirmed that reduced osteoblast frequency and enhanced Notch signaling were also features of the marrow stroma of human ALL tissues. Collectively, our findings suggest that therapeutically targeting the leukemia-infiltrated hematopoietic niche may restore HSPC homeostasis and improve the outcome of ALL patients. Cancer Res; 76(6); 1641–52. ©2016 AACR.

Supplementary Movie 2 from Simultaneous Visualization of Protumorigenic Src and MT1-MMP Activities with Fluorescence Resonance Energy Transfer

Invasion-promoting MT1-MMP is directly linked to tumorigenesis and metastasis. Our studies led us to identify those genes, the expression of which is universally linked to MT1-MMP in multiple tumor types. Genome-wide expression profiling of MT1-MMP–overexpressing versus MT1-MMP–silenced cancer cells and a further data mining analysis of the preexisting expression database of 190 human tumors of 14 cancer types led us to identify 11 genes, the expression of which correlated firmly and universally with that of MT1-MMP (P < 0.00001). These genes included regulators of energy metabolism (NNT), trafficking and membrane fusion (SLCO2A1 and ANXA7), signaling and transcription (NR3C1, JAG1, PI3Kδ, and CK2α), chromatin rearrangement (SMARCA1), cell division (STK38/NDR1), apoptosis (DAPK1), and mRNA splicing (SNRPB2). Our subsequent extensive analysis of cultured cells, tumor xenografts, and cancer patient biopsies supported our data mining. Our results suggest that transcriptional reprogramming of the specific downstream genes, which themselves are associated with tumorigenesis, represents a distinctive “molecular signature” of the proteolytically active MT1-MMP. We suggest that the transactivation activity of MT1-MMP contributes to the promigratory cell phenotype, which is induced by this tumorigenic proteinase. The activated downstream gene network then begins functioning in unison with MT1-MMP to rework the signaling, transport, cell division, energy metabolism, and other critical cell functions and to commit the cell to migration, invasion, and, consequently, tumorigenesis. [Cancer Res 2008;68(11):4086–96]

Supplementary Table 4 from Molecular Signature of MT1-MMP: Transactivation of the Downstream Universal Gene Network in Cancer

Supplementary Movie 1 from Simultaneous Visualization of Protumorigenic Src and MT1-MMP Activities with Fluorescence Resonance Energy Transfer

An elevated expression of membrane type-1 matrix metalloproteinase (MT1-MMP) is closely associated with multiple malignancies. Recently, we discovered that recycled MT1-MMP was trafficked along the tubulin cytoskeleton into the centrosomal compartment and cleaved the integral centrosomal protein pericentrin-2. These events correlated with the induction of chromosome instability and aneuploidy in nonmalignant Madine-Darby canine kidney cells. Accordingly, we hypothesized that MT1-MMP is an oncogene that promotes malignant transformation of normal cells rather than just an enzyme that supports growth of preexisting tumors. To prove our hypothesis, we transfected normal 184B5 human mammary epithelial cells with MT1-MMP (184B5-MT1 cells). MT1-MMP was colocalized with pericentrin in the centrosomal compartment and especially in the midbody of dividing cells. 184B5-MT1 cells acquired the ability to activate MMP-2, to cleave pericentrin, and to invade the Matrigel matrix. 184B5-MT1 cells exhibited aneuploidy, and they were efficient in generating tumors in the orthotopic xenograft model in immunodeficient mice. Because of the absence of tumor angiogenesis and the resulting insufficient blood supply, the tumors then regressed with significant accompanying necrosis. Gene array studies confirmed a significant up-regulation of oncogenes and tumorigenic genes but not the angiogenesis-promoting genes in 184B5-MT1 cells. We believe that our data point to a novel function of MT1-MMP in the initial stages of malignant transformation and to new and hitherto unknown transition mechanism from normalcy to malignancy. (Cancer Res 2006; 66(21): 10460-5)

Supplementary Figure 1 from Molecular Signature of MT1-MMP: Transactivation of the Downstream Universal Gene Network in Cancer

Supplementary Table 3 from Molecular Signature of MT1-MMP: Transactivation of the Downstream Universal Gene Network in Cancer

Supplementary Table 2 from Molecular Signature of MT1-MMP: Transactivation of the Downstream Universal Gene Network in Cancer