Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible SH2-containing (CIS) protein, a key negative regulator of interleukin (IL)-15 signaling, with fourth generation 'armored' chimeric antigen receptor (CAR-IL-15) engineering of cord blood (CB) derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell anti-tumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that targeting a cytokine checkpoint further enhances the antitumor activity of IL-15 secreting armored CAR-NK cells by promoting their metabolic fitness and anti-tumor activity. This combined approach represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.

Targeting the αv integrin-TGF-β axis improves natural killer cell function against glioblastoma stem cells Running title-GBM induce NK cell dysfunction via integrin-TGF- axis

Yap1 is a transcriptional co-activator of the Hippo pathway. The importance of Yap1 in early cell fate decision during embryogenesis has been well established, though its role in embryonic stem (ES) cells remains elusive. Here, we report that Yap1 plays crucial roles in normal differentiation rather than self-renewal of ES cells. Yap1-depleted ES cells maintain undifferentiated state with a typical colony morphology as well as robust alkaline phosphatase activity. These cells also retain comparable levels of the core pluripotent factors, such as Pou5f1 and Sox2, to the levels in wild-type ES cells without significant alteration of lineage-specific marker genes. Conversely, overexpression of Yap1 in ES cells promotes nuclear translocation of Yap1, resulting in disruption of self-renewal and triggering differentiation by up-regulating lineage-specific genes. Moreover, Yap1-deficient ES cells show impaired induction of lineage markers during differentiation. Collectively, our data demonstrate that Yap1 is a required factor for proper differentiation of mouse ES cells, while remaining dispensable for self-renewal.

Purpose: Natural killer (NK)-cell recognition and function against NK-resistant cancers remain substantial barriers to the broad application of NK-cell immunotherapy. Potential solutions include bispecific engagers that target NK-cell activity via an NK-activating receptor when simultaneously targeting a tumor-specific antigen, as well as enhancing functionality using IL12/15/18 cytokine pre-activation. Experimental Design: We assessed single-cell NK-cell responses stimulated by the tetravalent bispecific antibody AFM13 that binds CD30 on leukemia/lymphoma targets and CD16A on various types of NK cells using mass cytometry and cytotoxicity assays. The combination of AFM13 and IL12/15/18 pre-activation of blood and cord blood–derived NK cells was investigated in vitro and in vivo. Results: We found heterogeneity within AFM13-directed conventional blood NK cell (cNK) responses, as well as consistent AFM13-directed polyfunctional activation of mature NK cells across donors. NK-cell source also impacted the AFM13 response, with cNK cells from healthy donors exhibiting superior responses to those from patients with Hodgkin lymphoma. IL12/15/18-induced memory-like NK cells from peripheral blood exhibited enhanced killing of CD30+ lymphoma targets directed by AFM13, compared with cNK cells. Cord-blood NK cells preactivated with IL12/15/18 and ex vivo expanded with K562-based feeders also exhibited enhanced killing with AFM13 stimulation via upregulation of signaling pathways related to NK-cell effector function. AFM13–NK complex cells exhibited enhanced responses to CD30+ lymphomas in vitro and in vivo. Conclusions: We identify AFM13 as a promising combination with cytokine-activated adult blood or cord-blood NK cells to treat CD30+ hematologic malignancies, warranting clinical trials with these novel combinations.

No abstract

BMI1 is a component of the Polycomb Repressive Complex 1 (PRC1), which plays a key role in maintaining epigenetic silencing during development. BMI1 also participates in gene silencing during DNA damage response, but the precise downstream function of BMI1 in gene silencing is unclear. Here we identified the UBR5 E3 ligase as a downstream factor of BMI1. We found that UBR5 forms damage-inducible nuclear foci in a manner dependent on the PRC1 components BMI1, RNF1 (RING1a), and RNF2 (RING1b). Whereas transcription is repressed at UV-induced lesions on chromatin, depletion of the PRC1 members or UBR5 alone derepressed transcription elongation at these sites, suggesting that UBR5 functions in a linear pathway with PRC1 in inducing gene silencing at lesions. Mass spectrometry (MS) analysis revealed that UBR5 associates with BMI1 as well as FACT components SPT16 and SSRP1. We found that UBR5 localizes to the UV-induced lesions along with SPT16. We show that UBR5 ubiquitinates SPT16, and depletion of UBR5 or BMI1 leads to an enlargement of SPT16 foci size at UV lesions, suggesting that UBR5 and BMI1 repress SPT16 enrichment at the damaged sites. Consistently, depletion of the FACT components effectively reversed the transcriptional derepression incurred in the UBR5 and BMI1 KO cells. Finally, UBR5 and BMI1 KO cells are hypersensitive to UV, which supports the notion that faulty RNA synthesis at damaged sites is harmful to the cell fitness. Altogether, these results suggest that BMI1 and UBR5 repress the polymerase II (Pol II)-mediated transcription at damaged sites, by negatively regulating the FACT-dependent Pol II elongation.erturbation of chromatin structures can cause inappropriate gene expression and loss of genome integrity. Polycomb proteins are recognized in all metazoans for their conserved transcriptional repressive function. The canonical Polycomb Repressive Complex 1 (PRC1) contains BMI1, RNF1 (RING1a), RNF2 (RING1b), core components (PC), Polyhomeotic (PH), and CBX proteins (1, 2). BMI1 serves as a key regulatory component of the PRC1 complex, which is required to maintain the transcriptionally repressed state of many genes throughout development via chromatin remodeling and histone modification (1). The only known enzymatic activity of the BMI1-containing PRC1 complex is to monoubiquitinate histone H2A at Lys-119 (K119) residue, which is associated with transcriptional repression (1, 3, 4). However, the E3 ubiquitin ligase activity of RNF2 or the H2AK119-Ub is dispensable for repression of canonical PRC1 target genes during mouse or Drosophila embryonic development, respectively (5, 6), suggesting that the PRC1 complex may also induce gene silencing through other mechanisms (7). A series of studies has suggested that multiple distinct forms of the PRC1 complex with varying components could exist, and each of these may have distinct modes of regulation and functions (reviewed in ref. 2).In addition to its well-known role as an oncogene, recent evidence suggests that BMI1 participates in the DNA damage res...

MicroRNA expression profiling in human liver progenitor cells following hepatocytic differentiation identified miR-122 and miR-194 as the microRNAs most strongly upregulated during hepatocytic differentiation of progenitor cells. MiR-194 was also highly upregulated following hepatocytic differentiation of human embryonic stem cells (hESCs). Overexpression of miR-194 in progenitor cells accelerated their differentiation into hepatocytes, as measured by morphological features such as canaliculi and expression of hepatocytic markers. Overexpression of miR-194 in hESCs induced their spontaneous differentiation, a phenotype accompanied with accelerated loss of the pluripotent factors OCT4 and NANOG and decrease in mesoderm marker HAND1 expression. We then identified YAP1 as a direct target of miR-194. Inhibition of YAP1 strongly induced hepatocytic differentiation of progenitor cells and YAP1 over expression reversed the miR-194-induced hepatocytic differentiation of progenitor cells. In conclusion, we identified miR-194 as a potent inducer of hepatocytic differentiation of progenitor cells and further identified YAP1 as a mediator of miR-194's effects on hepatocytic differentiation and liver progenitor cell fate.

Adoptive cell therapy with viral-specific T cells has been successfully used to treat life-threatening viral infections, supporting the application of this approach against COVID-19. We expand SARS-CoV-2 T-cells from the peripheral blood of COVID-19-recovered donors and non-exposed controls using different culture conditions. We observe that the choice of cytokines modulates the expansion, phenotype and hierarchy of antigenic recognition by SARS-CoV-2 T-cells. Culture with IL-2/4/7 but not other cytokine-driven conditions result in >1000 fold expansion in SARS-CoV-2 T-cells with a retained phenotype, function and hierarchy of antigenic recognition when compared to baseline (pre-expansion) samples. Expanded CTLs are directed against structural SARS-CoV-2 proteins, including the receptor-binding domain of Spike. SARS-CoV-2 T-cells cannot be efficiently expanded from the peripheral blood of non-exposed controls. Since corticosteroids are used for the management of severe COVID-19, we propose an efficient strategy to inactivate the glucocorticoid receptor gene ( NR3C1 ) in SARS-CoV-2 CTLs using CRISPR-Cas9 gene editing.