Erratum: Development of a gene edited next-generation hematopoietic cell transplant to enable acute myeloid leukemia treatment by solving off-tumor toxicity.

[This corrects the article DOI: 10.1016/j.omtm.2023.101135.].

Development of a gene edited next-generation hematopoietic cell transplant to enable acute myeloid leukemia treatment by solving off-tumor toxicity.

Immunotherapy of acute myeloid leukemia (AML) has been challenging because the lack of tumor-specific antigens results in "on-target, off-tumor" toxicity. To unlock the full potential of AML therapies, we used CRISPR-Cas9 to genetically ablate the myeloid protein CD33 from healthy donor hematopoietic stem and progenitor cells (HSPCs), creating tremtelectogene empogeditemcel (trem-cel). Trem-cel is a HSPC transplant product designed to provide a reconstituted hematopoietic compartment that is resistant to anti-CD33 drug cytotoxicity. Here, we describe preclinical studies and process development of clinical-scale manufacturing of trem-cel. Preclinical data showed proof-of-concept with loss of CD33 surface protein and no impact on myeloid cell differentiation or function. At clinical scale, trem-cel could be manufactured reproducibly, routinely achieving >70% CD33 editing with no effect on cell viability, differentiation, and function. Trem-cel pharmacology studies using mouse xenograft models showed long-term engraftment, multilineage differentiation, and persistence of gene editing. Toxicology assessment revealed no adverse findings, and no significant or reproducible off-target editing events. Importantly, CD33-knockout myeloid cells were resistant to the CD33-targeted agent gemtuzumab ozogamicin in vitro and in vivo. These studies supported the initiation of the first-in-human, multicenter clinical trial evaluating the safety and efficacy of trem-cel in patients with AML (NCT04849910).

Loss of Grem1-lineage chondrogenic progenitor cells causes osteoarthritis.

Osteoarthritis (OA) is characterised by an irreversible degeneration of articular cartilage. Here we show that the BMP-antagonist Gremlin 1 (Grem1) marks a bipotent chondrogenic and osteogenic progenitor cell population within the articular surface. Notably, these progenitors are depleted by injury-induced OA and increasing age. OA is also caused by ablation of Grem1 cells in mice. Transcriptomic and functional analysis in mice found that articular surface Grem1-lineage cells are dependent on Foxo1 and ablation of Foxo1 in Grem1-lineage cells caused OA. FGFR3 signalling was confirmed as a promising therapeutic pathway by administration of pathway activator, FGF18, resulting in Grem1-lineage chondrocyte progenitor cell proliferation, increased cartilage thickness and reduced OA. These findings suggest that OA, in part, is caused by mechanical, developmental or age-related attrition of Grem1 expressing articular cartilage progenitor cells. These cells, and the FGFR3 signalling pathway that sustains them, may be effective future targets for biological management of OA.

Loss of Grem1-articular cartilage progenitor cells causes osteoarthritis.

Osteoarthritis (OA), which carries an enormous disease burden across the world, is characterised by irreversible degeneration of articular cartilage (AC), and subsequently bone. The cellular cause of OA is unknown. Here, using lineage tracing in mice, we show that the BMP-antagonist Gremlin 1 (Grem1) marks a novel chondrogenic progenitor (CP) cell population in the articular surface that generates joint cartilage and subchondral bone during development and adulthood. Notably, this CP population is depleted in injury-induced OA, and with age. OA is also induced by toxin-mediated ablation of Grem1 CP cells in young mice. Transcriptomic analysis and functional modelling in mice revealed articular surface Grem1-lineage cells are dependent on Foxo1; ablation of Foxo1 in Grem1-lineage cells led to early OA. This analysis identified FGFR3 signalling as a therapeutic target, and injection of its activator, FGF18, caused proliferation of Grem1-lineage CP cells, increased cartilage thickness, and reduced OA pathology. We propose that OA arises from the loss of CP cells at the articular surface secondary to an imbalance in progenitor cell homeostasis and present a new progenitor population as a locus for OA therapy.

Multiplex Base Editing to Protect from CD33-Directed Therapy: Implications for Immune and Gene Therapy.

On-target toxicity to normal cells is a major safety concern with targeted immune and gene therapies. Here, we developed a base editing (BE) approach exploiting a naturally occurring CD33 single nucleotide polymorphism leading to removal of full-length CD33 surface expression on edited cells. CD33 editing in human and nonhuman primate (NHP) hematopoietic stem and progenitor cells (HSPCs) protects from CD33-targeted therapeutics without affecting normal hematopoiesis in vivo , thus demonstrating potential for novel immunotherapies with reduced off-leukemia toxicity. For broader applications to gene therapies, we demonstrated highly efficient (>70%) multiplexed adenine base editing of the CD33 and gamma globin genes, resulting in long-term persistence of dual gene-edited cells with HbF reactivation in NHPs. In vitro , dual gene-edited cells could be enriched via treatment with the CD33 antibody-drug conjugate, gemtuzumab ozogamicin (GO). Together, our results highlight the potential of adenine base editors for improved immune and gene therapies.

Allogeneic cord blood regulatory T cells can resolve lung inflammation.

BACKGROUND AIMS: CD4+CD25+CD127lo regulatory T cells (Tregs) are responsible for maintaining immune homeostasis. Tregs can be rendered defective and deficient as a result of the immune imbalance seen in lung injury, and such dysfunction can play a major role in continued tissue inflammation. The authors hypothesized that adoptive therapy with healthy allogeneic umbilical cord blood (UCB)-derived Tregs may be able to resolve inflammation. RESULTS: Ex vivo-expanded UCB Tregs exhibited a unique phenotype with co-expression of CD45RA+CD45RO+ >80% and lung homing markers, including CD49d. UCB Tregs did not turn pathogenic when exposed to IL-6. Co-culture with increasing doses of dexamethasone led to a synergistic increase in UCB Treg-induced apoptosis of conventional T cells (Tcons), which translated into significantly higher suppression of proliferating Tcons, especially at a lower Treg:Tcon ratio. Multiple injections of UCB Tregs led to their preferential accumulation in lung tissue in an immune injury xenogenic model. A significant decrease in lung resident cytotoxic CD8+ T cells (P = 0.0218) correlated with a sustained decrease in their systemic distribution compared with controls (P < 0.0001) (n = 7 per arm) as well as a decrease in circulating human soluble CD40 ligand level (P = 0.031). Tissue architecture was preserved in the treatment arm, and a significant decrease in CD3+ and CD8+ burden was evident in immunohistochemistry analysis. CONCLUSIONS: UCB Treg adoptive therapy is a promising therapeutic strategy for treatment of lung injury.

A novel therapeutic bispecific format based on synthetic orthogonal heterodimers enables T cell activity against Acute myeloid leukemia.

Many therapeutic bispecific T-cell engagers (BiTEs) are in clinical trials. A modular and efficient process to create BiTEs would accelerate their development and clinical applicability. In this study, we present the design, production, and functional activity of a novel bispecific format utilizing synthetic orthogonal heterodimers to form a multichain modular design. Further addition of an immunoglobulin hinge region allowed a stable covalent linkage between the heterodimers. As proof-of-concept, we utilized CD33 and CD3 binding scFvs to engage leukemia cells and T-cells respectively. We provide evidence that this novel bispecific T-cell engager (termed IgGlue-BiTE) could bind both CD3+ and CD33+ cells and facilitates robust T-cell mediated cytotoxicity on AML cells in vitro. In a mouse model of minimal residual disease, we showed that the novel IgGlue-BiTE greatly extended survival, and mice of this treatment group were free of leukemia in the bone marrow. These findings suggest that the IgGlue-BiTE allows for robust simultaneous engagement with both antigens of interest in a manner conducive to T cell cytotoxicity against AML. These results suggest a compelling modular system for bispecific antibodies, as the CD3- and CD33-binding domains can be readily swapped with domains binding to other cancer- or immune cell-specific antigens.

Post-vaccination outcomes in association with four COVID-19 vaccines in the Kingdom of Bahrain.

With the emergence of new SARS-Cov2 variants, critical questions have arisen about: (1) the effectiveness of the available COVID-19 vaccines developed to protect against the original Wuhan (wild type) variant and (2) the magnitude and clinical consequences of post-vaccination infections in the context of the Delta variant of SARS-Cov2. While some "real world" experiences with various vaccines have been reported, to our knowledge, few have examined comparative outcomes of various vaccines in one country as new SARS-CoV-2 variants have emerged. Here we present an analysis of COVID-19 related outcomes from a national database in Bahrain, a country with a total population of 1.51 million, where four vaccines were deployed (total vaccinated = 1,003,960 adults): AstraZeneca (AZ/Covishield), Pfizer/BioNtech, Sinopharm and Sputnik V. We compare the four vaccines, based on the following post-vaccination outcomes: SARS-CoV-2 infections, hospitalisations, ICU admissions and deaths, compared to unvaccinated individuals. We conclude that the four vaccines used in Bahrain were effective in significantly reducing all four COVID-19 related outcomes compared to unvaccinated individuals, prior to, and during the period when the Delta variant predominated in the country. However, compared to the three other vaccines, individuals vaccinated with Sinopharm vaccine had a higher risk of post-vaccination infections, hospitalisations and ICU admissions (e.g., 6.94%, 2.24%, 1.99% and 1.52% of COVID-19 cases of Sinopharm, Sputnik V, Pfizer and Covishield recipients, respectively, required hospitalisation versus 13.66% of COVID-19 cases among unvaccinated individuals); however, given the confounding factors, this needs to be confirmed by further studies. We find no evidence of biased selection for any vaccine, but note waning protection of the Pfizer/BioNtech vaccine during the January to June 2021 period in the age > 60 y cohort; however, this cannot be distinguished from the overall fall in hospitalisations overall. Our findings support the value of vaccination in preventing COVID-19 related outcomes, provide real world estimates on the outcomes and frequencies of post-vaccination infections for the four vaccines, which may inform vaccine selection in the context of the Delta variant across the globe.

Subversion of Serotonin Receptor Signaling in Osteoblasts by Kynurenine Drives Acute Myeloid Leukemia.

Remodeling of the microenvironment by tumor cells can activate pathways that favor cancer growth. Molecular delineation and targeting of such malignant-cell nonautonomous pathways may help overcome resistance to targeted therapies. Herein we leverage genetic mouse models, patient-derived xenografts, and patient samples to show that acute myeloid leukemia (AML) exploits peripheral serotonin signaling to remodel the endosteal niche to its advantage. AML progression requires the presence of serotonin receptor 1B (HTR1B) in osteoblasts and is driven by AML-secreted kynurenine, which acts as an oncometabolite and HTR1B ligand. AML cells utilize kynurenine to induce a proinflammatory state in osteoblasts that, through the acute-phase protein serum amyloid A (SAA), acts in a positive feedback loop on leukemia cells by increasing expression of IDO1-the rate-limiting enzyme for kynurenine synthesis-thereby enabling AML progression. This leukemia-osteoblast cross-talk, conferred by the kynurenine-HTR1B-SAA-IDO1 axis, could be exploited as a niche-focused therapeutic approach against AML, opening new avenues for cancer treatment. SIGNIFICANCE: AML remains recalcitrant to treatments due to the emergence of resistant clones. We show a leukemia-cell nonautonomous progression mechanism that involves activation of a kynurenine-HTR1B-SAA-IDO1 axis between AML cells and osteoblasts. Targeting the niche by interrupting this axis can be pharmacologically harnessed to hamper AML progression and overcome therapy resistance. This article is highlighted in the In This Issue feature, p. 873.

SF3B1 mutant-induced missplicing of MAP3K7 causes anemia in myelodysplastic syndromes.

SF3B1 is the most frequently mutated RNA splicing factor in cancer, including in ∼25% of myelodysplastic syndromes (MDS) patients. SF3B1-mutated MDS, which is strongly associated with ringed sideroblast morphology, is characterized by ineffective erythropoiesis, leading to severe, often fatal anemia. However, functional evidence linking SF3B1 mutations to the anemia described in MDS patients harboring this genetic aberration is weak, and the underlying mechanism is completely unknown. Using isogenic SF3B1 WT and mutant cell lines, normal human CD34 cells, and MDS patient cells, we define a previously unrecognized role of the kinase MAP3K7, encoded by a known mutant SF3B1-targeted transcript, in controlling proper terminal erythroid differentiation, and show how MAP3K7 missplicing leads to the anemia characteristic of SF3B1-mutated MDS, although not to ringed sideroblast formation. We found that p38 MAPK is deactivated in SF3B1 mutant isogenic and patient cells and that MAP3K7 is an upstream positive effector of p38 MAPK. We demonstrate that disruption of this MAP3K7-p38 MAPK pathway leads to premature down-regulation of GATA1, a master regulator of erythroid differentiation, and that this is sufficient to trigger accelerated differentiation, erythroid hyperplasia, and ultimately apoptosis. Our findings thus define the mechanism leading to the severe anemia found in MDS patients harboring SF3B1 mutations.

The Origin and Contribution of Cancer-Associated Fibroblasts in Colorectal Carcinogenesis.

BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs) play an important role in colorectal cancer (CRC) progression and predict poor prognosis in CRC patients. However, the cellular origins of CAFs remain unknown, making it challenging to therapeutically target these cells. Here, we aimed to identify the origins and contribution of colorectal CAFs associated with poor prognosis. METHODS: To elucidate CAF origins, we used a colitis-associated CRC mouse model in 5 different fate-mapping mouse lines with 5-bromodeoxyuridine dosing. RNA sequencing of fluorescence-activated cell sorting-purified CRC CAFs was performed to identify a potential therapeutic target in CAFs. To examine the prognostic significance of the stromal target, CRC patient RNA sequencing data and tissue microarray were used. CRC organoids were injected into the colons of knockout mice to assess the mechanism by which the stromal gene contributes to colorectal tumorigenesis. RESULTS: Our lineage-tracing studies revealed that in CRC, many ACTA2+ CAFs emerge through proliferation from intestinal pericryptal leptin receptor (Lepr)+ cells. These Lepr-lineage CAFs, in turn, express melanoma cell adhesion molecule (MCAM), a CRC stroma-specific marker that we identified with the use of RNA sequencing. High MCAM expression induced by transforming growth factor ß was inversely associated with patient survival in human CRC. In mice, stromal Mcam knockout attenuated orthotopically injected colorectal tumoroid growth and improved survival through decreased tumor-associated macrophage recruitment. Mechanistically, fibroblast MCAM interacted with interleukin-1 receptor 1 to augment nuclear factor κB-IL34/CCL8 signaling that promotes macrophage chemotaxis. CONCLUSIONS: In colorectal carcinogenesis, pericryptal Lepr-lineage cells proliferate to generate MCAM+ CAFs that shape the tumor-promoting immune microenvironment. Preventing the expansion/differentiation of Lepr-lineage CAFs or inhibiting MCAM activity could be effective therapeutic approaches for CRC.

Assessing the Capability of Chemical Ameliorants to Reduce the Bioavailability of Heavy Metals in Bulk Fly Ash Contaminated Soil.

In-situ rehabilitation of fly ash at dumping sites has rarely been addressed for crop production due to growth-related constraints, largely of heavy metal (HM) contamination in soils and crops. Current communication deals with a novel approach to identify a suitable management option for rejuvenating the contaminated soils. In this background, a 60-days incubation experiment was conducted with different fly ash-soil mixtures (50 + 50%, A1; 75 + 25%, A2; 100 + 0%, A3) along with four ameliorants, namely, lime (T1), sodium sulphide (T2), di-ammonium phosphate (T3), and humic acid (T4) at 30 ± 2 °C to assess the ability of different fly ash-soil-ameliorant mixtures in reducing bio-availability of HMs. Diethylenetriaminepentaacetic acid (DTPA)-extractable bio-available HM contents for lead (Pb), cadmium (Cd), nickel (Ni), and chromium (Cr) and their respective ratios to total HM contents under the influence of different treatments were estimated at 0, 15, 30, 45, and 60 days of incubation. Further, the eco-toxicological impact of different treatments on soil microbial properties was studied after 60 days of experimentation. A1T1 significantly recorded the lowest bio-availability of HMs (~49-233% lower) followed by A2T1 (~35-133%) among the treatments. The principal component analysis also confirmed the superiority of A1T1 and A2T1 in this regard. Further, A1T1 achieved low contamination factor and ecological risk with substantial microbial biomass carbon load and dehydrogenase activity. Thus, liming to fly ash-soil mixture at 50:50 may be considered as the best management option for ameliorating metal toxicity. This technology may guide thermal power plants to provide the necessary package of practices for the stakeholders to revive their contaminated lands for better environmental sustainability.

Anomalous Diffusion and Lévy Walks Distinguish Active from Inertial Turbulence.

Bacterial swarms display intriguing dynamical states like active turbulence. Now, using a hydrodynamic model, we show that such dense active suspensions manifest superdiffusion, via Lévy walks, which masquerades as a crossover from ballistic to diffusive scaling in measurements of mean-squared displacements, and is tied to the emergence of hitherto undetected oscillatory streaks in the flow. Thus, while laying the theoretical framework of an emergent advantageous strategy in the collective behavior of microorganisms, our Letter underlines the essential differences between active and inertial turbulence.

Role of myeloid-derived suppressor cells in hormone-dependent cancers.

Tumour-infiltrating myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid cells. The main feature of MDSCs is their ability to suppress T-cell activation and function, which leads to immunosuppressive activity in the tumour microenvironment. Higher numbers of circulating and tumour-infiltrating MDSCs have been observed in a large number of patients with various types of tumour, and are linked to poor prognosis, especially in hormone-driven tumours. Recently, it has been demonstrated that the recruitment of MDSCs in prostate cancer confers resistance to canonical endocrine therapies, opening a new approach to the treatment of hormone-driven cancer patients.

Challenges and Solutions to Bringing Chimeric Antigen Receptor T-Cell Therapy to Myeloid Malignancies.

ABSTRACT: Myeloid malignancies including myelodysplastic syndromes and acute myeloid leukemia are a group of clonal hematopoietic stem progenitor cell disorders mainly effecting the elderly. Chemotherapeutic approaches improved the outcome in majority of the patients, but it is generally associated with severe toxicities and relapse and does not benefit all the patients. With the success of adoptive cell therapies including chimeric antigen receptor T-cell therapy in treating certain B-cell malignancies, these therapeutic approaches are also being tested for myeloid malignancies, but the preclinical and limited clinical trial data suggest there are significant challenges. The principal hurdle to efficient targeted immunotherapy approaches is the lack of a unique targetable antigen on cancer cells leading to off-target effects including myelosuppression due to depletion of normal myeloid cells. Advanced age of the patients, comorbidities, immunosuppressive bone marrow microenvironment, and cytokine release syndrome are some other challenges that are not unique to myeloid malignancies but pose significant challenge for the successful adaptation of this approach for treatment. In this review, we highlight the challenges and solutions to adopt chimeric antigen receptor T-cell therapies to treat myeloid malignancies.

Studies on leaching characteristics of electronic waste for metal recovery using inorganic and organic acids and base.

In this paper, we report leaching of precious and scattered metals such as gold (Au), copper (Cu), nickel (Ni), zinc (Zn), iron (Fe), and lead (Pb) from printed circuit boards of scrap mobile phones by hydrometallurgical process using inorganic acid, organic acid and base. The amount of metals leached by different leachants are quantified using atomic absorption spectroscopy. Among various inorganic acids, aqua regia (mixture of nitric acid (HNO3) and hydrochloric acid) is found to be the strongest leachant for most of the metals such as Zn (2.04 wt %), Fe (17.90 wt %), Ni (0.66 wt %), Pb (5.86 wt %) and Au (0.04 wt %). The basic leachant, ammonium thiosulphate is found to be very effective in leaching of Au (0.03125 wt %). The dissolution of Cu in HNO3 gives the highest amount of Cu in the solvent, that is, ∼ 7.52 wt %. The metallic phases present in the electronic waste before and after leaching are identified by X-ray diffraction analysis. The microscopic structure has been studied using a scanning electron microscope which depicts erosion of the structure after leaching.

Robust p53 Stabilization Is Dispensable for Its Activation and Tumor Suppressor Function.

p53 is a short-lived protein with low basal levels under normal homeostasis conditions. However, upon DNA damage, levels of p53 dramatically increase for its activation. Although robust stabilization of p53 serves as a "trademark" for DNA damage responses, the requirement for such dramatic protein stabilization in tumor suppression has not been well addressed. Here we generated a mutant p53KQ mouse where all the C-terminal domain lysine residues were mutated to glutamines (K to Q mutations at K367, K369, K370, K378, K379, K383, and K384) to mimic constitutive acetylation of the p53 C-terminus. Because of p53 activation, p53KQ/KQ mice were perinatal lethal, yet this lethality was averted in p53KQ/- mice, which displayed normal postnatal development. Nevertheless, p53KQ/- mice died prematurely due to anemia and hematopoiesis failure. Further analyses indicated that expression of the acetylation-mimicking p53 mutant in vivo induces activation of p53 targets in various tissues without obviously increasing p53 levels. In the well-established pancreatic ductal adenocarcinoma (PDAC) mouse model, expression of the acetylation-mimicking p53-mutant protein effectively suppressed K-Ras-induced PDAC development in the absence of robust p53 stabilization. Together, our results provide proof-of-principle evidence that p53-mediated transcriptional function and tumor suppression can be achieved independently of its robust stabilization and reveal an alternative approach to activate p53 function for therapeutic purposes. SIGNIFICANCE: Although robust p53 stabilization is critical for acute p53 responses such as DNA damage, this study underscores the important role of low basal p53 protein levels in p53 activation and tumor suppression.

The Balance of Stromal BMP Signaling Mediated by GREM1 and ISLR Drives Colorectal Carcinogenesis.

BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs), key constituents of the tumor microenvironment, either promote or restrain tumor growth. Attempts to therapeutically target CAFs have been hampered by our incomplete understanding of these functionally heterogeneous cells. Key growth factors in the intestinal epithelial niche, bone morphogenetic proteins (BMPs), also play a critical role in colorectal cancer (CRC) progression. However, the crucial proteins regulating stromal BMP balance and the potential application of BMP signaling to manage CRC remain largely unexplored. METHODS: Using human CRC RNA expression data, we identified CAF-specific factors involved in BMP signaling, then verified and characterized their expression in the CRC stroma by in situ hybridization. CRC tumoroids and a mouse model of CRC hepatic metastasis were used to test approaches to modify BMP signaling and treat CRC. RESULTS: We identified Grem1 and Islr as CAF-specific genes involved in BMP signaling. Functionally, GREM1 and ISLR acted to inhibit and promote BMP signaling, respectively. Grem1 and Islr marked distinct fibroblast subpopulations and were differentially regulated by transforming growth factor ß and FOXL1, providing an underlying mechanism to explain fibroblast biological dichotomy. In patients with CRC, high GREM1 and ISLR expression levels were associated with poor and favorable survival, respectively. A GREM1-neutralizing antibody or fibroblast Islr overexpression reduced CRC tumoroid growth and promoted Lgr5+ intestinal stem cell differentiation. Finally, adeno-associated virus 8 (AAV8)-mediated delivery of Islr to hepatocytes increased BMP signaling and improved survival in our mouse model of hepatic metastasis. CONCLUSIONS: Stromal BMP signaling predicts and modifies CRC progression and survival, and it can be therapeutically targeted by novel AAV-directed gene delivery to the liver.