ALBERT

Description: Recognition of DNA viruses, such as cytomegaloviruses (CMVs), through pattern-recognition receptor (PRR) pathways involving MyD88 or STING constitute a first-line defense against infections mainly through production of type I interferon (IFN-I). However, the role of these pathways in different tissues is incompletely understood, an issue particularly relevant to the CMVs which have broad tissue tropisms. Herein, we contrasted anti-viral effects of MyD88 versus STING in distinct cell types that are infected with murine CMV (MCMV). Bone marrow chimeras revealed STING-mediated MCMV control in hematological cells, similar to MyD88. However, unlike MyD88, STING also contributed to viral control in non-hematological, stromal cells. Infected splenic stromal cells produced IFN-I in a cGAS-STING-dependent and MyD88-independent manner, while we confirmed plasmacytoid dendritic cell IFN-I had inverse requirements. MCMV-induced natural killer cytotoxicity was dependent on MyD88 and STING. Thus, MyD88 and STING contribute to MCMV control in distinct cell types that initiate downstream immune responses.

Description: Chronic granulomatous disease (CGD) can result from any of four single gene defects involving the components of the superoxide (O−2 ) generating phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We show that transduction of peripheral blood CD34+ hematopoietic progenitors from a p67phox deficient CGD patient with replication defective amphotropic retrovirus encoding p67phox (MFGS-p67phox) significantly corrected the CGD functional defect in phagocyte oxidase activity in vitro. Using a chemiluminescence assay of oxidase activity, we showed that transduced patient CD34+ progenitors differentiating to myeloid cells in culture produced 25% of the total superoxide produced by normal CD34+ progenitors differentiating in culture. A flow cytometric assay of oxidase activity used to assess the oxidase function of individual cells in the cultures indicated that up to 32% of maturing granulocytes derived from transduced CD34+ progenitors from the p67phox CGD patient were oxidase positive with the average level of correction per granulocyte of 85% of that seen with granulocytes in similar cultures of CD34+ progenitors from normal volunteers. Nitroblue tetrazolium dye reduction assays of colonies of transduced progenitors in soft agar indicated that in some studies restoration of oxidase activity occurred in myeloid cells within 44% of granulocyte-erythrocyte-monocyte colonies, and within 28% of the combined group of granulocyte colonies/monocyte colonies/granulocyte-monocyte colonies. These high correction rates were achieved without any selective regimen to enrich for transduced cells. This study provides a basis for development of gene therapy for the p67phox deficient form of CGD.

Description: Chronic granulomatous disease (CGD) can result from any of four single gene defects involving the components of the superoxide (O−2 ) generating phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We show that transduction of peripheral blood CD34+ hematopoietic progenitors from a p67phox deficient CGD patient with replication defective amphotropic retrovirus encoding p67phox (MFGS-p67phox) significantly corrected the CGD functional defect in phagocyte oxidase activity in vitro. Using a chemiluminescence assay of oxidase activity, we showed that transduced patient CD34+ progenitors differentiating to myeloid cells in culture produced 25% of the total superoxide produced by normal CD34+ progenitors differentiating in culture. A flow cytometric assay of oxidase activity used to assess the oxidase function of individual cells in the cultures indicated that up to 32% of maturing granulocytes derived from transduced CD34+ progenitors from the p67phox CGD patient were oxidase positive with the average level of correction per granulocyte of 85% of that seen with granulocytes in similar cultures of CD34+ progenitors from normal volunteers. Nitroblue tetrazolium dye reduction assays of colonies of transduced progenitors in soft agar indicated that in some studies restoration of oxidase activity occurred in myeloid cells within 44% of granulocyte-erythrocyte-monocyte colonies, and within 28% of the combined group of granulocyte colonies/monocyte colonies/granulocyte-monocyte colonies. These high correction rates were achieved without any selective regimen to enrich for transduced cells. This study provides a basis for development of gene therapy for the p67phox deficient form of CGD.

Description: Selection for a promiscuous enzyme activity provides substantial opportunity for competition between endogenous and newly-encountered substrates to influence the evolutionary trajectory, an aspect that is often overlooked in laboratory directed evolution studies. We selected the Escherichia coli nitro/quinone reductase NfsA for chloramphenicol detoxification by simultaneously randomising eight active site residues and interrogating ~250,000,000 reconfigured variants. Analysis of every possible intermediate of the two best chloramphenicol reductases revealed complex epistatic interactions. In both cases, improved chloramphenicol detoxification was only observed after an R225 substitution that largely eliminated activity with endogenous quinones. Error-prone PCR mutagenesis reinforced the importance of R225 substitutions, found in 100% of selected variants. This strong activity trade-off demonstrates that endogenous cellular metabolites hold considerable potential to shape evolutionary outcomes. Unselected prodrug-converting activities were mostly unaffected, emphasising the importance of negative selection to effect enzyme specialisation, and offering an application for the evolved genes as dual-purpose selectable/counter-selectable markers.

Description: Background: Insights into molecular biology and signaling pathways have led to the development of innovative therapeutic strategies to treat B-cell malignacies. In R/R CLL, targeted agents including Bruton tyrosine kinase inhibitors (BTKi) and B-cell lymphoma 2 inhibitors (Bcl-2i) have emerged as new standard of care (Ghia, 2019; Seymour, 2018). More recently, the combination of these two agents has shown impressive and synergistic clinical results with significantly higher complete response (CR) rates reported compared with either agent alone (Jain, 2019). This combination leverages the ability of BTKi to mobilize and egress malignant B-cells from the protective tumor microenvironment and to disrupt cell adhesion-mediated resistance; thereby increasing B-cell sensitivity to novel pro-apoptotic mechanisms (e.g. Bcl-2i). KRT-232 is a potent and highly selective second-generation inhibitor of MDM2 which rapidly activates p53 to induce apoptosis in malignant hematopoetic stem cells and progenitor cells. Studies in CLL and DLBCL cell lines showed MDM2i overcame Bcl-2 overexpression leading to induction of apoptosis through the upregulation of p21, BAX, and PUMA and downregulation of Bcl-xL (Drakos, 2011; Ciardullo, 2019). As such, targeting the p53-driven apoptotic pathway may be a novel mechanism, independent of Bcl-2 inhibition, to treat B-cell malignacies. KRT-232-driven apoptosis requires tumor cells to express the wild-type encoding p53 gene (TP53WT) found in ~60% and ~80% of pts with R/R CLL and R/R DLBCL, respectively (Miao, 2019; Lazarian, 2017). Acalabrutinib is a potent and highly selective second-generation inhibitor of BTK with established efficacy in R/R CLL and an improved safety profile (Ghia, 2020). In pts with R/R DLBCL, acalabrutinib monotherapy was well tolerated and demonstrated promising clinical activity (24% overall response rate [ORR], 19% CR; Dyer 2018). Aberrant signaling through BTK drives malignant B-cell proliferation, trafficking and sequestering into protective niches causing lymphadenopathy. BTK signaling plays a critical role in CXCR4-CXCL12 driven chemotaxis and integrin-mediated adhesion. Stromal interactions within the protective microevironment have been associated with aberrant B-cell proliferation, treatment resistance and protection against apoptosis (Montresor, 2018). The combinination of KRT-232, a novel cell-death pathway agent, with acalabrutinib, a selective and potent cell trafficking modulator, has the potential to demonstrate synergistic clinical activity across a broad range of B-cell malignancies with a differentiated safety profile compared with similar targeted combinations. To this effect, nonclinical synergy has been demonstrated with combinations of MDM2i and BTKi in CLL (Voltan, 2016) and DLBCL (Gu, 2019). Study Design and Methods: KRT-232 + acalabrutinib is being evaluated in a Phase 1b-2 open-label, global, multicenter study in pts with R/R TP53WT DLBCL (Cohort 1) or R/R TP53WT CLL (Cohort 2; Figure). Pts aged ≥18 years with histologically confirmed disease and ECOG PS of 0-2 will be enrolled. Pts with known CNS involvement, prior exposure to MDM2i or BTKi and recent prior therapy (anticancer treatment

Description: Natural killer (NK) cells are essential for early protection against virus infection and must metabolically adapt to the energy demands of activation. Here, we found upregulation of the metabolic adaptor hypoxia-inducible factor-1α (HIF1α) is a feature of mouse NK cells during murine cytomegalovirus (MCMV) infection in vivo. HIF1α-deficient NK cells failed to control viral load, causing increased morbidity. No defects were found in effector functions of HIF1αKO NK cells; however, their numbers were significantly reduced. Loss of HIF1α did not affect NK cell proliferation during in vivo infection and in vitro cytokine stimulation. Instead, we found that HIF1α-deficient NK cells showed increased expression of the pro-apoptotic protein Bim and glucose metabolism was impaired during cytokine stimulation in vitro. Similarly, during MCMV infection HIF1α-deficient NK cells upregulated Bim and had increased caspase activity. Thus, NK cells require HIF1α-dependent metabolic functions to repress Bim expression and sustain cell numbers for an optimal virus response.