Alpha-tocopherol (vitamin E) is a plant-derived dietary lipid that is essential for the health of most animals, including humans. Originally discovered as a fertility factor in rodents, the primary health-promoting properties of the vitamin in humans was shown to be protection of neuromuscular functions. Heritable vitamin E deficiency manifests in spinocerebellar ataxia that can be stabilized by timely supplementation with high-dose α-tocopherol. The molecular basis for α-tocopherol's biological activities has been attributed primarily to the vitamin's efficacy in preventing lipid peroxidation in membranes and lipoproteins, but the possibility that the vitamin possesses additional biological activities has been postulated and debated in the literature without conclusive resolution. We designed and synthesized a novel analog of α-tocopherol, 6-hydroxymethyl α-tocopherol (6-HMTC), which retains most of the vitamin's structural, physical, and biochemical properties, yet lacks measurable radical-trapping antioxidant activity. 6-HMTC bound to the tocopherol transfer protein with high (nanomolar) affinity, like that of the natural vitamin, attesting to the analog's preservation of structural integrity. Yet, 6-HMTC did not inhibit lipid peroxidation or associated ferroptotic cell death. Notably, 6-HMTC modulated the expression of some genes in a manner essentially identical to that exhibited by α-tocopherol. These findings support the notion that α-tocopherol modulates gene expression via an antioxidant-independent mechanism.

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.

Background: Chemoresistance is an existing challenge faced in the treatment of the hairy cell leukemia variant (HCL-v). Classical hairy cell leukemia (HCL-c) is very sensitive to the standard of care with purine nucleoside analogs (PNAs) cladribine (cDa) and pentostatin. However, almost half of these patients eventually become less sensitive to chemotherapy and relapse. HCL-variant (HCL-v) is a biologically distinct entity from HCL-c that is not sensitive to frontline PNA therapy, and this treatment is not recommended for these patients. To address these treatment challenges, we investigated the role of B-cell activating factor (BAFF) in promoting HCL-v cell chemoresistance. Methods: Flow cytometry and quantitative PCR were used to measure the levels of BAFF and its receptors. To determine BAFF activated pathways in HCL-c and HCL-v, the Bonna-12 HCL-c cell line or HCL-v patient-derived cancer cells were stimulated with recombinat BAFF and activation of common BAFF-activated pathways, including the nonclassical nuclear factor kappa B (NF-κB) pathway, the Extracellular Signal-Regulated Kinase (Erk) and phosphatidylinositol-3 (PI-3) kinase (PI3K)/AKT serine/threonine kinase (AKT) pathways were measured by western blotting. To test whether BAFF signaling promotes chemoresistance in HCL-v, we stimulated patient-derived HCL-v cells with BAFF and performed RNA sequencing. Lastly, to confirm the functional implications of BAFF signaling in HCL-v, we treated patient-derived HCL-v cells with exogenous BAFF before treatment with cladribine. Results: We found that HCL-v patient-derived cancer cells express receptors of BAFF at varying degrees and express relatively lower levels of membrane-bound BAFF ligand expression. BAFF stimulation of these cells resulted in substantial activation of the nonclassical NF-κB pathway, which is known to promote anti-apoptotic and pro-survival effects in B-cell cancers. Conversely, in the Bonna-12 cell line, we observed constitutive activation of the nonclassical NF-κB pathway. Through RNA sequencing, we found that BAFF upregulates a myriad of genes that are known to promote chemoresistance in various cancers, including IL1, CXCL1/2, CXCL5, CXCL8, TRAF3, and PTGS2. Lastly, we found that BAFF protects these cells from cladribine-induced cell death in vitro. Conclusions: We conclude that BAFF provides chemo-protection in HCL-v cells by activating nonclassical NF-κB signaling, which results in the upregulation of multiple pro-survival or anti-apoptotic genes. Our results highlight an important role of BAFF in HCL-v resistance to chemotherapy and suggest that the BAFF blockade may enhance the chemosensitivity to PNAs in drug-resistant HCL-v patients.

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.

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.

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.