A genome-scale in vivo loss-of-function screen identifies Phf6 as a lineage-specific regulator of leukemia cell growth.

We performed a genome-scale shRNA screen for modulators of B-cell leukemia progression in vivo. Results from this work revealed dramatic distinctions between the relative effects of shRNAs on the growth of tumor cells in culture versus in their native microenvironment. Specifically, we identified many "context-specific" regulators of leukemia development. These included the gene encoding the zinc finger protein Phf6. While inactivating mutations in PHF6 are commonly observed in human myeloid and T-cell malignancies, we found that Phf6 suppression in B-cell malignancies impairs tumor progression. Thus, Phf6 is a "lineage-specific" cancer gene that plays opposing roles in developmentally distinct hematopoietic malignancies.

Heterologous Vaccination and Checkpoint Blockade Synergize To Induce Antileukemia Immunity.

Checkpoint blockade-based immunotherapies are effective in cancers with high numbers of nonsynonymous mutations. In contrast, current paradigms suggest that such approaches will be ineffective in cancers with few nonsynonymous mutations. To examine this issue, we made use of a murine model of BCR-ABL(+) B-lineage acute lymphoblastic leukemia. Using a principal component analysis, we found that robust MHC class II expression, coupled with appropriate costimulation, correlated with lower leukemic burden. We next assessed whether checkpoint blockade or therapeutic vaccination could improve survival in mice with pre-established leukemia. Consistent with the low mutation load in our leukemia model, we found that checkpoint blockade alone had only modest effects on survival. In contrast, robust heterologous vaccination with a peptide derived from the BCR-ABL fusion (BAp), a key driver mutation, generated a small population of mice that survived long-term. Checkpoint blockade strongly synergized with heterologous vaccination to enhance overall survival in mice with leukemia. Enhanced survival did not correlate with numbers of BAp:I-A(b)-specific T cells, but rather with increased expression of IL-10, IL-17, and granzyme B and decreased expression of programmed death 1 on these cells. Our findings demonstrate that vaccination to key driver mutations cooperates with checkpoint blockade and allows for immune control of cancers with low nonsynonymous mutation loads.

The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers.

Colorectal tumours that are wild type for KRAS are often sensitive to EGFR blockade, but almost always develop resistance within several months of initiating therapy. The mechanisms underlying this acquired resistance to anti-EGFR antibodies are largely unknown. This situation is in marked contrast to that of small-molecule targeted agents, such as inhibitors of ABL, EGFR, BRAF and MEK, in which mutations in the genes encoding the protein targets render the tumours resistant to the effects of the drugs. The simplest hypothesis to account for the development of resistance to EGFR blockade is that rare cells with KRAS mutations pre-exist at low levels in tumours with ostensibly wild-type KRAS genes. Although this hypothesis would seem readily testable, there is no evidence in pre-clinical models to support it, nor is there data from patients. To test this hypothesis, we determined whether mutant KRAS DNA could be detected in the circulation of 28 patients receiving monotherapy with panitumumab, a therapeutic anti-EGFR antibody. We found that 9 out of 24 (38%) patients whose tumours were initially KRAS wild type developed detectable mutations in KRAS in their sera, three of which developed multiple different KRAS mutations. The appearance of these mutations was very consistent, generally occurring between 5 and 6 months following treatment. Mathematical modelling indicated that the mutations were present in expanded subclones before the initiation of panitumumab treatment. These results suggest that the emergence of KRAS mutations is a mediator of acquired resistance to EGFR blockade and that these mutations can be detected in a non-invasive manner. They explain why solid tumours develop resistance to targeted therapies in a highly reproducible fashion.

Adaptive Immunity to Leukemia Is Inhibited by Cross-Reactive Induced Regulatory T Cells.

BCR-ABL(+) acute lymphoblastic leukemia patients have transient responses to current therapies. However, the fusion of BCR to ABL generates a potential leukemia-specific Ag that could be a target for immunotherapy. We demonstrate that the immune system can limit BCR-ABL(+) leukemia progression although ultimately this immune response fails. To address how BCR-ABL(+) leukemia escapes immune surveillance, we developed a peptide: MHC class II tetramer that labels endogenous BCR-ABL-specific CD4(+) T cells. Naive mice harbored a small population of BCR-ABL-specific T cells that proliferated modestly upon immunization. The small number of naive BCR-ABL-specific T cells was due to negative selection in the thymus, which depleted BCR-ABL-specific T cells. Consistent with this observation, we saw that BCR-ABL-specific T cells were cross-reactive with an endogenous peptide derived from ABL. Despite this cross-reactivity, the remaining population of BCR-ABL reactive T cells proliferated upon immunization with the BCR-ABL fusion peptide and adjuvant. In response to BCR-ABL(+) leukemia, BCR-ABL-specific T cells proliferated and converted into regulatory T (Treg) cells, a process that was dependent on cross-reactivity with self-antigen, TGF-ß1, and MHC class II Ag presentation by leukemic cells. Treg cells were critical for leukemia progression in C57BL/6 mice, as transient Treg cell ablation led to extended survival of leukemic mice. Thus, BCR-ABL(+) leukemia actively suppresses antileukemia immune responses by converting cross-reactive leukemia-specific T cells into Treg cells.

Requirement for antiapoptotic MCL-1 in the survival of BCR-ABL B-lineage acute lymphoblastic leukemia.

The response of Philadelphia chromosome (Ph(+)) acute lymphoblastic leukemia (ALL) to treatment by BCR-ABL tyrosine kinase inhibitors (TKIs) has been disappointing, often resulting in short remissions typified by rapid outgrowth of drug-resistant clones. Therefore, new treatments are needed to improve outcomes for Ph(+) ALL patients. In a mouse model of Ph(+) B-lineage ALL, MCL-1 expression is dysregulated by the BCR-ABL oncofusion protein, and TKI treatment results in loss of MCL-1 expression prior to the induction of apoptosis, suggesting that MCL-1 may be an essential prosurvival molecule. To test this hypothesis, we developed a mouse model in which conditional allele(s) of Mcl-1 can be deleted either during leukemia transformation or later after the establishment of leukemia. We report that endogenous MCL-1's antiapoptotic activity promotes survival during BCR-ABL transformation and in established BCR-ABL(+) leukemia. This requirement for MCL-1 can be overcome by overexpression of other antiapoptotic molecules. We further demonstrate that strategies to inhibit MCL-1 expression potentiate the proapoptotic action of BCL-2 inhibitors in both mouse and human BCR-ABL(+) leukemia cell lines. Thus, strategies focused on antagonizing MCL-1 function and expression would be predicted to be effective therapeutic strategies.

Host thiopurine methyltransferase status affects mercaptopurine antileukemic effectiveness in a murine model.

BACKGROUND: Thiopurines are used for many cancers, including acute lymphoblastic leukemia (ALL). Patients with an inherited host defect in thiopurine methyltransferase (TPMT) are at high risk for life-threatening toxicity if treated with conventional dosages, but the impact on antileukemic efficacy is less clear. MATERIALS AND METHODS: We treated thiopurine-sensitive BCR-ABL+Arf-null Tpmt+/+ ALL in Tpmt+/+, +/-, or -/- recipient mice to test the impact of the host polymorphism on antileukemic efficacy. RESULTS: Median survival was similar in untreated mice of different Tpmt genotypes (16-18 days). However, in mice treated with low-dose mercaptopurine (such as tolerated by TPMT-/- patients), the difference in 30-day leukemia-free survival by Tpmt genotype was profound: 5% (±9%) for Tpmt+/+ mice, 47% (±26%) for Tpmt+/- mice, and 85% (±14%) for Tpmt-/- mice (P=5×10), indicating a substantial impact of host Tpmt status on thiopurine effectiveness. Among Tpmt+/+ recipient mice, leukemia-free survival improved with higher doses of mercaptopurine (similar to doses tolerated by wild-type patients) compared with lower doses, and at higher doses was comparable (P=0.6) to the survival of Tpmt-/- mice treated with the lower dose. CONCLUSIONS: These findings support the notion that germline polymorphisms in Tpmt affect not only host tissue toxicity but also antitumor effectiveness.

Chemotherapeutic agents circumvent emergence of dasatinib-resistant BCR-ABL kinase mutations in a precise mouse model of Philadelphia chromosome-positive acute lymphoblastic leukemia.

The introduction of cultured p185(BCR-ABL)-expressing (p185+) Arf (-/-) pre-B cells into healthy syngeneic mice induces aggressive acute lymphoblastic leukemia (ALL) that genetically and phenotypically mimics the human disease. We adapted this high-throughput Philadelphia chromosome-positive (Ph(+)) ALL animal model for in vivo luminescent imaging to investigate disease progression, targeted therapeutic response, and ALL relapse in living mice. Mice bearing high leukemic burdens (simulating human Ph(+) ALL at diagnosis) entered remission on maximally intensive, twice-daily dasatinib therapy, but invariably relapsed with disseminated and/or central nervous system disease. Although relapse was frequently accompanied by the eventual appearance of leukemic clones harboring BCR-ABL kinase domain (KD) mutations that confer drug resistance, their clonal emergence required prolonged dasatinib exposure. KD P-loop mutations predominated in mice receiving less intensive therapy, whereas high-dose treatment selected for T315I "gatekeeper" mutations resistant to all 3 Food and Drug Administration-approved BCR-ABL kinase inhibitors. The addition of dexamethasone and/or L-asparaginase to reduced-intensity dasatinib therapy improved long-term survival of the majority of mice that received all 3 drugs. Although non-tumor-cell-autonomous mechanisms can prevent full eradication of dasatinib-refractory ALL in this clinically relevant model, the emergence of resistance to BCR-ABL kinase inhibitors can be effectively circumvented by the addition of "conventional" chemotherapeutic agents with alternate antileukemic mechanisms of action.

A mammalian functional-genetic approach to characterizing cancer therapeutics.

Identifying mechanisms of drug action remains a fundamental impediment to the development and effective use of chemotherapeutics. Here we describe an RNA interference (RNAi)-based strategy to characterize small-molecule function in mammalian cells. By examining the response of cells expressing short hairpin RNAs (shRNAs) to a diverse selection of chemotherapeutics, we could generate a functional shRNA signature that was able to accurately group drugs into established biochemical modes of action. This, in turn, provided a diversely sampled reference set for high-resolution prediction of mechanisms of action for poorly characterized small molecules. We could further reduce the predictive shRNA target set to as few as eight genes and, by using a newly derived probability-based nearest-neighbors approach, could extend the predictive power of this shRNA set to characterize additional drug categories. Thus, a focused shRNA phenotypic signature can provide a highly sensitive and tractable approach for characterizing new anticancer drugs.

Correction: Synthesis of lead-free Cs3Sb2Br9 perovskite alternative nanocrystals with enhanced photocatalytic CO2 reduction activity.

Correction for 'Synthesis of lead-free Cs3Sb2Br9 perovskite alternative nanocrystals with enhanced photocatalytic CO2 reduction activity' by Chang Lu et al., Nanoscale, 2020, 12, 2987-2991, https://doi.org/10.1039/C9NR07722G.

Stage-specific Arf tumor suppression in Notch1-induced T-cell acute lymphoblastic leukemia.

Frequent hallmarks of T-cell acute lymphoblastic leukemia (T-ALL) include aberrant NOTCH signaling and deletion of the CDKN2A locus, which contains 2 closely linked tumor suppressor genes (INK4A and ARF). When bone marrow cells or thymocytes transduced with a vector encoding the constitutively activated intracellular domain of Notch1 (ICN1) are expanded ex vivo under conditions that support T-cell development, cultured progenitors rapidly induce CD4+/CD8+ T-ALLs after infusion into healthy syngeneic mice. Under these conditions, enforced ICN1 expression also drives formation of T-ALLs in unconditioned CD-1 nude mice, bypassing any requirements for thymic maturation. Retention of Arf had relatively modest activity in suppressing the formation of T-ALLs arising from bone marrow-derived ICN1+ progenitors in which the locus is epigenetically silenced, and all resulting Arf (+/+) tumors failed to express the p19(Arf) protein. In striking contrast, retention of Arf in thymocyte-derived ICN1+ donor cells significantly delayed disease onset and suppressed the penetrance of T-ALL. Use of cultured thymocyte-derived donor cells expressing a functionally null Arf-GFP knock-in allele confirmed that ICN1 signaling can induce Arf expression in vivo. Arf activation by ICN1 in T cells thereby provides stage-specific tumor suppression but also a strong selective pressure for deletion of the locus in T-ALL.

Whole-body physiologically based pharmacokinetic model for nutlin-3a in mice after intravenous and oral administration.

Nutlin-3a is an MDM2 inhibitor that is under investigation in preclinical models for a variety of pediatric malignancies, including retinoblastoma, rhabdomyosarcoma, neuroblastoma, and leukemia. We used physiologically based pharmacokinetic (PBPK) modeling to characterize the disposition of nutlin-3a in the mouse. Plasma protein binding and blood partitioning were assessed by in vitro studies. After intravenous (10 and 20 mg/kg) and oral (50, 100, and 200 mg/kg) dosing, tissue concentrations of nutlin-3a were determined in plasma, liver, spleen, intestine, muscle, lung, adipose, bone marrow, adrenal gland, brain, retina, and vitreous fluid. The PBPK model was simultaneously fit to all pharmacokinetic data using NONMEM. Nutlin-3a exhibited nonlinear binding to murine plasma proteins, with the unbound fraction ranging from 0.7 to 11.8%. Nutlin-3a disposition was characterized by rapid absorption with peak plasma concentrations at approximately 2 h and biphasic elimination consistent with a saturable clearance process. The final PBPK model successfully described the plasma and tissue disposition of nutlin-3a. Simulations suggested high bioavailability, rapid attainment of steady state, and little accumulation when administered once or twice daily at dosages up to 400 mg/kg. The final model was used to perform simulations of unbound tissue concentrations to determine which dosing regimens are appropriate for preclinical models of several pediatric malignancies.

Altered toxicological endpoints in humans from common quaternary ammonium compound disinfectant exposure.

Humans are frequently exposed to Quaternary Ammonium Compounds (QACs). QACs are ubiquitously used in medical settings, restaurants, and homes as cleaners and disinfectants. Despite their prevalence, nothing is known about the health effects associated with chronic low-level exposure. Chronic QAC toxicity, only recently identified in mice, resulted in developmental, reproductive, and immune dysfunction. Cell based studies indicate increased inflammation, decreased mitochondrial function, and disruption of cholesterol synthesis. If these findings translate to human toxicity, multiple physiological processes could be affected. This study tested whether QAC concentrations could be detected in the blood of 43 human volunteers, and whether QAC concentrations influenced markers of inflammation, mitochondrial function, and cholesterol synthesis. QAC concentrations were detected in 80 % of study participants. Blood QACs were associated with increase in inflammatory cytokines, decreased mitochondrial function, and disruption of cholesterol homeostasis in a dose dependent manner. This is the first study to measure QACs in human blood, and also the first to demonstrate statistically significant relationships between blood QAC and meaningful health related biomarkers. Additionally, the results are timely in light of the increased QAC disinfectant exposure occurring due to the SARS-CoV-2 pandemic. MAIN FINDINGS: This study found that 80 % of study participants contained QACs in their blood; and that markers of inflammation, mitochondrial function, and sterol homeostasis varied with blood QAC concentration.

Perovskite Quantum-Dot-in-Host for Detection of Ionizing Radiation.

The concept of quantum-dot-in-perovskite solids pioneered by Ning and co-workers introduces a useful class of solution-processed type I heterostructures for optoelectronics applications. Concurrent searches for solution-processable detectors of ionizing radiation have focused on lead-halide perovskites. As described in this issue of ACS Nano, Cao et al. examined CsPbBr3 nanocrystals imbedded in Cs4PbBr6 as a wider gap host and determined its performance and possibilities as a scintillator for X-ray imaging. In this Perspective, we describe issues and research opportunities on ionizing radiation imaging and spectroscopy based on the CsPbBr3@Cs4PbBr6 composite and other perovskite-dot-in-host combinations in which the dot may be of lower dimensionality than 3, and we explore ionizing radiation detectors using halide perovskites.

Synthesis of lead-free Cs3Sb2Br9 perovskite alternative nanocrystals with enhanced photocatalytic CO2 reduction activity.

A synthetic method for uniform and pure Cs3Sb2Br9 NCs has been developed. Cs3Sb2Br9 NCs exhibit a 10-fold increase in activity for the photocatalytic CO2 reduction reaction compared to CsPbBr3 NCs, achieving 510 µmol CO g-1 cat. after 4 h. Density functional theory shows that Cs3Sb2Br9 surfaces sufficiently expose Sb to allow reactivity, as opposed to the unreactive CsPbBr3 surface.

pH-Responsive branched polymernanoparticles.

We describe a new one-pot, single-step route for the preparation of pH-responsive branched polymer nanoparticles. These polymers, which are based on the pH-responsive monomer 2-(diethylamino)ethyl methacrylate (DEA) and hydrophilic macromonomer poly(ethyleneglycol) methacrylate (PEGMA), are synthesised using a modified conventional free-radical polymerisation. Consequently, their preparation is generic, scaleable and tolerant of functionality. In aqueous solution the branched copolymers form core-shell structures at basic pH and on reducing the solution pH they become hydrated and swell, displaying similar characteristics to those of pH-responsive shell cross-linked micelles and microgels. We demonstrate good control over the hydrodynamic particle size, polymer chain-end, and the uptake and release of a model hydrophobe and also the ability to tune the apparent pKa of the DEA residues by varying the degree of branching. These results augur well for commercially viable tunable release applications.

High throughput synthesis of polyesters using entropically driven ring-opening polymerizations.

Copolyesters were synthesized in a high throughput (HT) manner and in high yield on ca. a 90 mg scale using entropically driven ring-opening polymerizations (ED-ROPs). This synthetic approach is a valuable addition to the HT polymer synthesis arsenal in that it allows condensation-type polymers with relatively large repeat units, such as those in poly(ethylene terephthalate) and poly(butylene terephthalate), to be obtained easily. The synthetic procedure involved taking mixtures of the appropriate macrocyclic oligoesters and heating them together under neat conditions at 250-300 degrees C for 2 h in the presence of 0.1 mol % of di- n-butyltin oxide or tetra- n-butylammonium tetrafluoroborate. In most cases Mw values were >25,000 and, as expected for ED-ROPs, the polydispersity indices were close to 2.0. Higher molecular weights could be obtained by using longer reaction times, but this might lead to product decomposition. The method worked well for esters formally derived from aliphatic or aromatic acids and alcohols, but less well for esters derived from phenols. Attempts were also made to synthesize copolymers by mixing together the two homopolymers and heating with a catalyst. These reactions were successful in a few instances, but generally, they were not. This is probably because the homopolymers did not mix well. An aluminum reaction block with 36 wells lined with Teflon cups, that fitted snugly in a cylindrical Buchi oven, was the most successful method for carrying out syntheses in an HT manner.

The ARF tumor suppressor in acute leukemias: insights from mouse models of Bcr-Abl-induced acute lymphoblastic leukemia.

The prototypical Bcr-Abl chimeric oncoprotein is central to the pathogenesis of chronic myelogenous leukemias (CMLs) and a subset of acute lymphoblastic leukemias (Ph+ ALLs). The constitutive tyrosine kinase transforms either hematopoietic stem cells (in CML) or committed pre-B lymphoid progenitors (in Ph+ ALL) to generate these distinct diseases. The INK4A/ARF tumor suppressor locus is frequently deleted in both B- and T-lineage ALLs, including Ph+ ALL, whereas the locus remains intact in CML. In murine bone marrow transplant models and after transfer of syngeneic Bcr-Abl-transformed pre-B cells into immunocompetent recipient animals, Arf gene inactivation dramatically decreases the latency and enhances the aggressiveness of Bcr-Abl-induced lymphoblastic leukemia. Targeted inhibition of the Bcr-Abl kinase with imatinib provides highly effective therapy for CML, but Ph+ ALL patients do not experience durable remissions. Despite exquisite in vitro sensitivity of Arf-null, BCR-ABL+ pre-B cells to imatinib, these cells efficiently establish lethal leukemias when introduced into immunocompetent mice that receive continuous, maximal imatinib therapy. Bcr-Abl confers interleukin-7 (IL-7) independence to pre-B cells, but imatinib treatment restores the requirement for this cytokine. Hence, IL-7 can reduce the sensitivity of Bcr-Abl+ pre-B cells to imatinib. Selective inhibitors of both Bcr-Abl and the IL-7 transducing JAK kinases may therefore prove beneficial in treating Ph+ ALL.

Imbedded Nanocrystals of CsPbBr3 in Cs4 PbBr6 : Kinetics, Enhanced Oscillator Strength, and Application in Light-Emitting Diodes.

Solution-grown films of CsPbBr3 nanocrystals imbedded in Cs4 PbBr6 are incorporated as the recombination layer in light-emitting diode (LED) structures. The kinetics at high carrier density of pure (extended) CsPbBr3 and the nanoinclusion composite are measured and analyzed, indicating second-order kinetics in extended and mainly first-order kinetics in the confined CsPbBr3 , respectively. Analysis of absorption strength of this all-perovskite, all-inorganic imbedded nanocrystal composite relative to pure CsPbBr3 indicates enhanced oscillator strength consistent with earlier published attribution of the sub-nanosecond exciton radiative lifetime in nanoprecipitates of CsPbBr3 in melt-grown CsBr host crystals and CsPbBr3 evaporated films.

Ubiquitination of, and sumoylation by, the Arf tumor suppressor.

The Ink4a-Arf locus, which encodes two distinct tumor suppressor proteins, is inactivated in many cancers. Whereas p16Ink4a is an inhibitor of cyclin D-dependent kinases, p19Arf (p14ARF in humans) antagonizes the E3 ubiquitin protein ligase activity of Mdm2 to activate p53. We now recognize that Arf functions in both p53-dependent and -independent modes to counteract hyper-proliferative signals originating from proto-oncogene activation, but its p53-independent activities remain poorly understood. Arf proteins are highly basic (> 20% arginine content, pl > 12) and predominantly localize within nucleoli in physical association with an abundant acidic protein, nucleophosmin (NPM/B23). When bound to NPM, Arf proteins are relatively stable with half-lives of 6-8 hours. Although mouse p19Arf contains only a single lysine residue and human p14ARF has none, both proteins are N-terminally ubiquitinated and degraded in proteasomes. Through as yet uncharacterized mechanisms, p19Arf induces p53-independent sumoylation of a variety of cellular target proteins with which it interacts, including both Mdm2 and NPM. A naturally occurring NPM mutant (NPMc) expressed in myeloid leukemia cells redirects both wild-type NPM and p19Arf to the cytoplasm, inhibits Arf-induced sumoylation, and attenuates p53 activity. Thus, ubiquitination and sumoylation can each influence Arf tumor suppressor activity.

Stromal interaction molecule 1 (STIM1), a transmembrane protein with growth suppressor activity, contains an extracellular SAM domain modified by N-linked glycosylation.

Stromal interaction molecule 1 (STIM1) is a cell surface transmembrane glycoprotein implicated in tumour growth control and stromal-haematopoietic cell interactions. A single sterile alpha motif (SAM) protein-protein interaction domain is modelled within its extracellular region, a subcellular localisation not previously described for other SAM domain-containing proteins. We have defined the transmembrane topology of STIM1 by determining the sites of N-linked glycosylation. We have confirmed that STIM1 is modified by N-linked glycosylation at two sites within the SAM domain itself, deduced as asparagine residues N131 and N171, demonstrating that STIM1 is translocated across the membrane of the endoplasmic reticulum such that the SAM domain resides within the endoplasmic reticulum (ER) lumen. Both N-linked oligosaccharides remain endoglycosidase H-sensitive, indicating absence of full processing within the ER and Golgi. This immature modification is nevertheless sufficient and critical for cell surface expression of STIM1. We show that STIM1-STIM1 homotypic interactions are mediated via the cytoplasmic rather than the extracellular region of STIM1, excluding an essential role for the SAM domain in these protein interactions. These studies provide the first evidence for an extracellular localisation of a SAM domain within any protein, and the first example of a SAM domain modified by N-linked glycosylation.