Immunocompromised Mice Research Articles

The Role of over-Expressed β Globin in Driving Relapsed B - Cell Acute Lymphoblastic Leukemia (B-ALL)

Background: The prognosis for patients with relapsed acute lymphoblastic leukemia (ALL) remains suboptimal despite intensification of chemotherapy, immunotherapy and stem cell transplant. We and others have identified genetic and epigenetic alterations enriched at relapse that drive drug resistance and relapse. In our recent work mapping the epigenetic landscape of B-ALL, we discovered novel super-enhancers (SEs), or regions with increased H3K27 acetylation, enriched at relapse in a majority of patients, implicating a role in clonal evolution. One of these SEs was upstream of the β globin locus and lead to aberrant expression of hemoglobin beta subunit ( HBB) mRNA. HBB upregulation at relapse was also found in a large meta-analysis of gene expression data from pediatric diagnosis/relapse pairs. Of note, b globin is normally found in erythrocytes that interacts with other subunits to form functional hemoglobin. We hypothesized that HBB upregulation in blasts plays a role in relapse and aimed to determine whether it leads to drug tolerance, an early recovery phenotype and/or increased clonal potential. Methods: B-lineage REH, RS4;11 and 697 ALL cell lines were transduced with lentiviral vectors overexpressing either wild-type (WT) HBB or an empty vector (EV) control. Real time PCR was performed to confirm RNA expression and Western Blot analysis was performed to detect protein expression. We generated additional cell lines with a mutation in the translation start codon and another leading to a premature stop codon to unequivocally interrupt protein expression. The generated cell lines were then plated and evaluated for differences in apoptosis via Annexin V assay and proliferation, in response to chemotherapeutic agents commonly used in the treatment of ALL. Clonogenic growth in vitro was also assessed using MethoCult TM media. To test the possibility that HBB acted in generating a persistor cell phenotype, cell lines were placed in mercaptopurine and prednisone for 10-14 days at two different concentrations and counted throughout drug exposure and for an additional 10-14 days following removal of drug. Results: We did not observe significant differences in chemosensitivity to various chemotherapies used in the treatment of B-ALL. However, we did observe less cells undergoing apoptosis in HBB expressing cells compared to control following prolonged treatment with 6-mercaptopurine, suggesting an increase in a small population of drug tolerant cells. However, this did not result in a difference in recovery time following removal of the drug. We also demonstrated that β globin overexpression led to increased clonal growth in methylcellulose compared to control (Fig. 1A, REH) and this observation was replicated in a second cell line (Fig. 1B, RS4;11). While we confirmed mRNA expression, we were not able to confirm protein expression by Western blot or by immunoprecipitation. We therefore hypothesized that b globin mRNA, rather than protein, mediates the observed increase in clonal growth. Interestingly, we observed the same phenotype of increased clonal growth (Fig. 1) in mutated cell lines, suggesting a catalytic role for β globin RNA in expanding a leukemia stem cell population. Conclusions: The increased clonal potential upon overexpression of HBB could be indicative of the ability to reconstitute a tumor from a small subpopulation following treatment. Additionally, our data suggests that protein is not required for increased clonal growth leading us to hypothesize that catalytic RNA is responsible. We are also currently analyzing RNAseq data from the cell lines and will compare differential gene expression between the empty vector and HBB expressing cells to discover downstream pathways impacted by HBB. We will be testing this hypothesis further by performing limiting dilution analysis in an immunocompromised mouse model (with David Teachey, Children's Hospital of Philadelphia).

DIALing-up the preclinical characterization of gene-modified adoptive cellular immunotherapies.

The preclinical characterization of gene modified adoptive cellular immunotherapy candidates for clinical development often requires the use of mouse models. Gene-modified lymphocytes (GML) incorporating chimeric antigen receptors (CAR) and T-cell receptors (TCR) into immune effector cells require in vivo characterization of biological activity, mechanism of action, and preclinical safety. Typically, this characterization involves the assessment of dose-dependent, on-target, on-tumor activity in severely immunocompromised mice. While suitable for the purpose of evaluating T cell-expressed transgene function in a living host, this approach falls short in translating cellular therapy efficacy, safety, and persistence from preclinical models to humans. To comprehensively characterize cell therapy products in mice, we have developed a framework called "DIAL". This framework aims to enable an end-to-end understanding of genetically engineered cellular immunotherapies in vivo, from infusion to tumor clearance and long-term immunosurveillance. The acronym DIAL stands for Distribution, Infiltration, Accumulation, and Longevity, compartmentalizing the systemic attributes of gene-modified cellular therapy and providing a platform for optimization with the ultimate goal of improving therapeutic efficacy. This review will discuss both existent and emerging examples of DIAL characterization in mouse models, as well as opportunities for future development and optimization.

The effect of an exopolysaccharide probiotic molecule from Bacillus subtilis on breast cancer cells

IntroductionMany well-known risk factors for breast cancer are associated with dysbiosis (an aberrant microbiome). However, how bacterial products modulate cancer are poorly understood. In this study, we investigated the effect of an exopolysaccharide (EPS) produced by the commensal bacterium Bacillus subtilis on breast cancer phenotypes. Although B. subtilis is commonly included in probiotic preparations and its EPS protects against inflammatory diseases, it was virtually unknown whether B. subtilis-derived EPS affects cancer.MethodsThis work investigated effects of EPS on phenotypes of breast cancer cells as a cancer model. The phenotypes included proliferation, mammosphere formation, cell migration, and tumor growth in two immune compromised mouse models. RNA sequencing was performed on RNA from four breast cancer cells treated with PBS or EPS. IKKβ or STAT1 signaling was assessed using pharmacologic or RNAi-mediated knock down approaches. ResultsShort-term treatment with EPS inhibited proliferation of certain breast cancer cells (T47D, MDA-MB-468, HCC1428, MDA-MB-453) while having little effect on others (MCF-7, MDA-MB-231, BT549, ZR-75-30). EPS induced G1/G0 cell cycle arrest of T47D cells while increasing apoptosis of MDA-MB-468 cells. EPS also enhanced aggressive phenotypes in T47D cells including cell migration and cancer stem cell survival. Long-term treatment with EPS (months) led to resistance in vitro and promoted tumor growth in immunocompromised mice. RNA-sequence analysis showed that EPS increased expression of pro-inflammatory pathways including STAT1 and NF-κB. IKKβ and/or STAT1 signaling was necessary for EPS to modulate phenotypes of EPS sensitive breast cancer cells. DiscussionThese results demonstrate a multifaceted role for an EPS molecule secreted by the probiotic bacterium B. subtilis on breast cancer cell phenotypes. These results warrant future studies in immune competent mice and different cancer models to fully understand potential benefits and/or side effects of long-term use of probiotics.

Shifting the Antibody-Drug Conjugate Paradigm: A Trastuzumab-Gold-Based Conjugate Demonstrates High Efficacy against Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer Mouse Model.

Antibody-drug conjugates (ADCs) combine the selectivity of monoclonal antibodies (mAbs) with the efficacy of chemotherapeutics to target cancers without toxicity to normal tissue. Clinically, most chemotherapeutic ADCs are based on complex organic molecules, while the conjugation of metallodrugs to mAbs has been overlooked, despite the resurgent interest in metal-based drugs as cancer chemotherapeutics. In 2019, we described the first gold ADCs containing gold-triphenylphosphane fragments as a proof of concept. The ADCs (based on the antibody trastuzumab) were selective and highly active against HER2-positive breast cancer cells. In this study, we developed site-specific ADCs (Thio-1b and Thio-2b) using the cysteine-engineered trastuzumab derivative THIOMAB antibody technology with gold(I)-containing phosphanes and a maleimide-based linker amenable to bioconjugation (1b and 2b). In addition, we developed lysine-directed ADCs with gold payloads based on phosphanes and N-heterocyclic carbenes featuring an activated ester moiety (2c and 5c) with trastuzumab (Tras-2c and Tras-5c) and another anti-HER2 antibody, pertuzumab (Per-2c and Per-5c). Both sets of ADCs demonstrated significant anticancer potency in vitro assays. Based on these results, one ADC (Tras-2c), containing the [Au(PEt3)] fragment present in FDA-approved auranofin, was selected for an in vivo antitumor efficacy study. Immunocompromised mice xenografted with the HER2-positive human cancer cell line SKBR-3 exhibited almost complete tumor reduction and low toxicity with intravenous administration of Tras-2c. With this highly selective targeting system, we demonstrated that a subnanomolar cytotoxicity profile in cells is not required for an impressive antitumor effect in a mouse xenograft model.

Glutamine metabolism inhibition has dual immunomodulatory and antibacterial activities against Mycobacterium tuberculosis

As one of the most successful human pathogens, Mycobacterium tuberculosis (Mtb) has evolved a diverse array of determinants to subvert host immunity and alter host metabolic patterns. However, the mechanisms of pathogen interference with host metabolism remain poorly understood. Here we show that a glutamine metabolism antagonist, JHU083, inhibits Mtb proliferation in vitro and in vivo. JHU083-treated mice exhibit weight gain, improved survival, a 2.5 log lower lung bacillary burden at 35 days post-infection, and reduced lung pathology. JHU083 treatment also initiates earlier T-cell recruitment, increased proinflammatory myeloid cell infiltration, and a reduced frequency of immunosuppressive myeloid cells when compared to uninfected and rifampin-treated controls. Metabolomic analysis of lungs from JHU083-treated Mtb-infected mice reveals citrulline accumulation, suggesting elevated nitric oxide (NO) synthesis, and lowered levels of quinolinic acid which is derived from the immunosuppressive metabolite kynurenine. JHU083-treated macrophages also produce more NO potentiating their antibacterial activity. When tested in an immunocompromised mouse model of Mtb infection, JHU083 loses its therapeutic efficacy suggesting the drug’s host-directed effects are likely to be predominant. Collectively, these data reveal that JHU083-mediated glutamine metabolism inhibition results in dual antibacterial and host-directed activity against tuberculosis.

PAX3-FOXO1 dictates myogenic reprogramming and rhabdomyosarcoma identity in endothelial progenitors

Fusion-positive rhabdomyosarcoma (FP-RMS) driven by the expression of the PAX3-FOXO1 (P3F) fusion oncoprotein is an aggressive subtype of pediatric rhabdomyosarcoma. FP-RMS histologically resembles developing muscle yet occurs throughout the body in areas devoid of skeletal muscle highlighting that FP-RMS is not derived from an exclusively myogenic cell of origin. Here we demonstrate that P3F reprograms mouse and human endothelial progenitors to FP-RMS. We show that P3F expression in aP2-Cre expressing cells reprograms endothelial progenitors to functional myogenic stem cells capable of regenerating injured muscle fibers. Further, we describe a FP-RMS mouse model driven by P3F expression and Cdkn2a loss in endothelial cells. Additionally, we show that P3F expression in TP53-null human iPSCs blocks endothelial-directed differentiation and guides cells to become myogenic cells that form FP-RMS tumors in immunocompromised mice. Together these findings demonstrate that FP-RMS can originate from aberrant development of non-myogenic cells driven by P3F.

TMIC-35. TUMOR CELL-DERIVED SPERMIDINE ACCELERATES GLIOBLASTOMA PROGRESSION VIA LYMPHOCYTE DEPLETION

Abstract Therapeutic resistance in glioblastoma (GBM) is partly due to a highly immunosuppressive environment resulting in ineffective anti-tumor immune response. This likely underlies the lack of success of immunotherapies that are successful in other cancers; novel ways to target this environment are needed. Naturally occurring polyamines (PAs) have been identified as a putative therapeutic target in other cancers based on their increased presence and correlation to poorer prognosis. Under normal conditions, spermidine (SPD) – a member of the PA family – is critical for cell growth. While PAs are increased in GBM patients, little is known about their impact on GBM growth. In syngeneic immunocompetent mouse glioma models, mass spectrometry revealed that SPD increases in the tumor microenvironment and circulation as compared to non-tumor-bearing mice. We treated the same models with exogenous SPD, which significantly decreased survival. However, in immunocompromised mice, no survival difference was observed, suggesting SPD drives GBM progression through immune interaction. Immune profiling the mouse models treated with SPD showed peripheral reduction of CD4+ and CD8+ T cells. Due to the observed T cell changes, we repeated the exogenous SPD paradigm in RAG1 knockout mice that do not have circulating B/ T cells. Similar to NSG mice, we see no survival difference, indicating that SPD drives GBM by affecting lymphoid cells. To investigate the origin of SPD in our models, we knocked down the rate limiting enzyme of the PA biosynthesis pathway in glioma cells in immunocompetent mice. Mice implanted with knockdown cells showed a robust survival extension, suggesting that tumor cells produce SPD which affects the tumor microenvironment. Ongoing studies are evaluating uptake from the environment on the progression of GBM using pharmacological inhibitors. Understanding the interactions of PAs and the immune response as a whole will allow us to alter the environment to enhance immunotherapeutic efficacy.

Antitumor Effect by Either FLASH or Conventional Dose Rate Irradiation Involves Equivalent Immune Responses

Purpose: The capability of ultra-high dose rate FLASH radiotherapy (FLASH) to generate the FLASH effect has opened the possibility to enhance the therapeutic index of radiotherapy. The contribution of the immune response has frequently been hypothesized to account for a certain fraction of the anti-tumor efficacy and tumor kill of FLASH but has yet to be rigorously evaluated. Methods: To investigate the immune response as a potentially important mechanism of the anti-tumor effect of FLASH, various murine tumor models were grafted either subcutaneously or orthotopically into immunocompetent mice, or in moderately and severely immunocompromised mice. Mice were locally irradiated with single dose (20 Gy) or hypo-fractionated regimens (3 × 8 Gy; 2 × 6 Gy), using FLASH (≥ 2000 Gy/s) and Conventional dose rates (0.1 Gy/s, CONV), with/without anti-CTLA-4. Tumor growth was monitored over time, and immune profiling performed. Results: 20 Gy FLASH and CONV were isoeffective in delaying tumor growth in immunocompetent and moderately immunodeficient hosts, and increased tumor doubling time to >14 days vs. >7 days in control animals. Similar observations were obtained with hypo-fractionated scheme, regardless the microenvironment (subcutaneous flank vs. ortho lungs). Interestingly, in profoundly immunocompromised mice, 20 Gy FLASH retained anti-tumor activity and significantly increased tumor doubling time to >14 days vs. >8 days in control animals, suggesting a possible anti-tumor mechanism independent of the immune response. Analysis of the tumor microenvironment showed similar immune profiles after both irradiation modalities with significant decrease of lymphoid cells by ∼40 % and a corresponding increase of myeloid cells. In addition, FLASH and CONV did not increase TGF-β1 levels in tumors as compared to unirradiated control animals. Furthermore, when a complete and long-lasting anti-tumor response was obtained (> 140 days), both modalities of irradiation were able to generate a long-term immunological memory response. Conclusions: The present results clearly document that the tumor responses across multiple immunocompetent and immunodeficient mouse models are largely dose rate independent, and simultaneously contradict a major role of the immune response in the anti-tumor efficacy of FLASH. Therefore, our study indicates that FLASH is as potent as CONV in modulating anti-tumor immune response and can be used as an immunomodulatory agent.

IMMU-35. G-AMINOBUTYRIC ACID DRIVES GLIOBLASTOMA IN FEMALES BY ACTIVATING THE COMPLEMENT SYSTEM

Abstract Interaction between tumor and neural cells drives the progression of glioblastoma (GBM), which is characterized by enhanced immunosuppressive myeloid cell accumulation. Our previous results revealed that myeloid-derived suppressor cell subsets (MDSCs) play a central role in tumorigenesis in a sex-specific manner, with monocytic MDSCs (mMDSCs) accumulating in male tumors and granulocytic MDSCs (gMDSCs) correlating with a worse outcome in females. However, host factors governing sex differences in MDSC subset activity remained unknown. Here, we report that g-aminobutyric acid (GABA) receptor expression by gMDSCs informs their sex-specific behavior. We observed that only female gMDSCs but not male gMDSCs or mMDSCs from either sex respond to GABA by upregulating the antiviral innate immune pathway. As part of this signature, GABA-stimulated female gMDSCs had higher complement 1qa (C1qa) expression. To test the potential role of the GABA pathway on GBM outcome, we tested the GABA analogue pregabalin in preclinical models. Female mice treated with pregabalin succumbed to disease earlier, consistent with our previous observation that gMDSCs have a central role in GBM progression in females. In contrast, pregabalin treatment had no effect in male mice with tumors or immunocompromised female mice. To further evaluate whether C1q is a relevant physiological target, we performed single-cell sequencing of tumor-infiltrating versus circulating MDSC subsets and detected high levels of C1q expression in gMDSCs from tumors. C1q KO transgenic female mice had significantly better outcomes compared to wild-type littermates upon implantation with syngeneic mouse GBM lines, while C1q expression status did not impact the outcome in males. Correspondingly, only in female patients C1q expression correlated with poor prognosis. Collectively, these results identify a neuron-immune-cancer communication axis and indicate that GABA and C1q alter anti-tumor immune responses in a sex-specific manner. These results support the future assessment of the GABA pathway and complement inhibitors as potential immunotherapy agents.

IMMU-04. LOW-INTENSITY PULSED ULTRASOUND ENHANCES CAR T CELL THERAPY BY INDUCING TWO-WAY IMMUNE CELL TRAFFICKING – T CELL INTO TUMOR AND MYELOID CELL INTO PERIPHERY IN A SYNGENEIC MOUSE GLIOBLASTOMA MODEL

Abstract Chimeric Antigen Receptor (CAR) T-cell therapy has been successful against hematological malignancies. However, it has not established efficacy in glioblastoma (GBM) patients. Significant challenges currently limiting the effectiveness of CAR T-cell therapy for patients with GBM include poor CAR-T cell trafficking to GBM due to the blood-brain barrier, and the presence of highly immunosuppressive and pro-tumoral tumor-associated macrophages (TAMs) in the tumor microenvironment. Furthermore, the heterogeneous expression of target antigens in GBM can lead to antigen escape and therapeutic resistance. Low-intensity pulsed ultrasound with microbubbles (LIPU/MB) has been shown to enhance CAR T-cell trafficking and improve survival in immunocompromised xenograft mice GBM models. In this study, we investigated whether multiple rounds of LIPU/MB can improve not only CAR T-cell but also endogenous T-cell infiltration into the tumors and induce myeloid cell trafficking into lymph nodes (LNs), thus enhancing CAR T-cell therapy against GBM in the immunocompetent syngeneic mice model. We recently developed a novel immunocompetent transgenic (Tg) mouse that expresses an anti-EGFRvIII CAR on T-cells and enables us to investigate the interaction between CAR T-cells and the endogenous immune environment. C57BL6/J mice bearing intracerebral EGFRvIII+ GBM received an intravenous administration of anti-EGFRvIII CAR T-cells derived from Tg-mice. The mice were stratified to receive single-LIPU (1 min before CAR T-cells administration) or triple-LIPU/MB (1 min before CAR T-cells administration, then two and four days later). Triple-LIPU/MB significantly suppressed tumor growth and improved survival and was associated with increased CAR T-cells and endogenous T-cells infiltration into the tumors compared with single-LIPU/MB or without LIPU. Furthermore, draining cervical LNs showed increased macrophages trafficking after triple-LIPU/MB. Moreover, TAMs isolated from triple-LIPU/MB mice brains showed higher levels of MHC-II expression. These results suggest that LIPU/MB may promote the tumor antigen presentation to endogenous T-cells and enhance the efficacy of CAR T-cell therapy for GBM.

Immune system humanization in dyslipidemic Rag2-KO/IL2rg-KO/CD47-KO/LDL-R KO mice controls atherosclerosis

Abstract Background and aim Given the key role of the immune response during atherosclerosis and the therapeutic interest of biologics targeting human immune cells, the need of experimental models to translate molecular mechanisms and to test therapeutic approaches for atherosclerosis is continuously increasing. Here we describe the characteristics of an innovative immunodeficient mouse humanized with hCD34+ cells on an atheroprone background. Methods LDLR-KO mice were crossed with the immunodeficient C57BL/6J strain Rag2-KO/IL2rg-KO/CD47-KO (TKO, IMSR_JAX:025730) to generate an immunocompromised dyslipidemic mouse recipient TKO-LDLR KO of human hematopoietic stem cells (hCD34+). Results TKO-LDLR KO were first characterized for their immune and metabolic profile. TKO mice are deficient in mature lymphocytes and NK cells and this profile was conserved in TKO-LDLR KO mice. Under high cholesterol diet for 8 weeks, both male and females TKO-LDLR KO present monocytosis with increased levels of Ly6Chi monocytes compared to TKO-LDLR KO at standard diet, develop marked dyslipidemia (total cholesterol 870.9 and 890.1 mg/dL male and females respectively), steatosis and atherosclerosis. This profile confirms the suitability of TKO-LDLR KO mice for atherosclerosis studies. Next we tested the impact of immune system humanization on atherosclerosis. TKO-LDLR KO pups received a low-dose irradiation (150-200 cGy) and thereafter 1,5-2 x 10^5 hCD34+ were injected with in the liver. Engraftment of human leukocytes (hCD45+) was evaluated after two months by flow cytometry analysis from tail blood. This approach allows to reconstitute between 10-30% of hCD45+, mainly B and T cells. The humanization with hCD34+ cells affected atherosclerosis development as compared to non-humanized TKO-LDLR KO mice. Conclusion We have generated and characterized for the first time a humanized dyslipidemic TKO-LDLR KO mice. This mouse model presents human B and T cells and represent and important toll to investigate the impact of biologics targeted toward human targets in the context of atherosclerosis.

Engineering Sequential Mutations into Human HSPCs Yields an Aggressive Myeloid Malignancy Allowing for Interrogation of Preleukemic Transformation

Introduction: Acute Myeloid Leukemia (AML) is an aggressive myeloid malignancy that develops as the result of stepwise accumulation of mutations in hematopoietic stem and progenitor cells (HSPCs). Multiple studies of the clonal evolution of AML have determined that in general, the order of mutation acquisition follows a stereotypical fashion: mutations that occur early in development of AML involve epigenetic modifiers, whereas mutations that occur later more commonly affect proteins involved in signaling and proliferation. While multiple studies have demonstrated the ability of various combinations of mutations to promote leukemic transformation in mouse models, the majority of this work has studied concurrent alteration of driver genes, which may not accurately reflect human disease biology. To this end, we aimed to generate a model of AML by sequentially introducing mutations into human HSPCs to study driver gene dependent and independent pathways in preleukemic transformation. Methods: Our model of sequential mutation acquisition in human AML employs CRISPR/Cas9-mediated gene disruption and AAV6-mediated homology directed repair (HDR) in human cord blood CD34+ HSPCs, allowing for high-efficiency knockout of epigenetic modifiers ( DNMT3A or TET2) and expression of an activating signaling protein ( NRAS G12D) along with the BFP fluorescent marker. Mutations are introduced sequentially through a serial xenotransplantation approach in immunocompromised mice, allowing for stepwise characterization of the preleukemic and transformed states of disease. Results: To establish a model of preleukemic transformation using human HSPCs, we first developed a double synthetic guide RNA (sgRNA) knockout strategy that produces a ~55-65 bp deletion in either DNMT3A or TET2, leading to highly efficient INDEL (insertion or deletion) generation in cord blood CD34+ HSPCs and effective protein knockdown. Fourteen weeks after transplantation into immunocompromised mice, DNMT3A-KO, TET2-KO, and control cells demonstrate high levels of bi-lineage bone marrow engraftment (60-90%) and greater than 95% INDEL frequency at targeted loci. DNMT3A-KO, TET2-KO, and control CD34+ cells harvested from primary transplantation are subsequently edited to express NRAS G12D-BFP at efficiencies similar to freshly isolated cells (10-20%), and sorted BFP+ cells successfully engraft into secondary recipient mice. Consistent with observations from genetically engineered mouse models, we found that the combination of either DNMT3A-KO or TET2-KO with NRAS G12D in human cord blood CD34+ cells results in a rapidly fatal high-grade myeloid neoplasm in mice. Moribund mice develop peripheral cytopenias, decreased body weight, enlarged spleens, and have infiltration of CD45+ human cells in bone marrow, spleen, and liver. Human cells isolated from bone marrow retain near 100% BFP positivity and show persistence of >95% DNMT3A-KOor TET2-KO INDELs. BFP+ cells display a monocytic immunophenotype (CD34-, CD33+, CD14 variable, CD16 variable) and have a monocytic appearance on H&E staining. Experiments introducing TET2-KO/ NRAS G12D mutations using either a sequential or concurrent approach demonstrate mutational synergy between TET2-KO and NRAS G12Dwhen compared with NRAS G12D alone. Comparisons of disease kinetics and phenotype when TET2-KO and NRAS G12D mutations are introduced sequentially vs concurrently are currently underway to investigate how temporal dynamics in epigenetic changes after TET2-KO affect subsequent transformation with NRAS G12D. In addition, genome-wide DNA methylation and gene expression characterization from cells in preleukemic ( DNMT3A-KO and TET2-KO alone) and transformed states ( DNMT3A-KO/ NRAS G12D and TET2-KO/ NRAS G12D) are ongoing to explore mechanisms underlying these observations. Conclusion: Our work demonstrates that human HSPCs can be efficiently engineered to model the combination of early epigenetic modifier loss with expression of an activating signaling mutation, as is commonly seen in AML, resulting in a fully penetrant, high-grade myeloid neoplasm. To our knowledge, this represents the first sequential model of preleukemic transformation in genetically engineered human cells. Importantly, the sequential nature of our approach allows for manipulation of the preleukemic phase of disease and subsequent interrogation of myeloid transformation.

Tetraspanin CD81 Supports Cancer Stem Cell Function and Represents a Therapeutic Vulnerability in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease associated with a poor outcome, necessitating aggressive therapies to achieve remission. A major contributing factor to treatment resistance and disease relapse is the presence of leukemic stem cells (LSC). Although challenging, targeting LSC constitutes an imperative approach to improve patient outcomes and limit side effects. In a previous study, we have uncovered a prognostic association between CD81 cell surface overexpression and worse outcome for AML patients. In the present work, we validated that CD81 overexpression of either protein or mRNA in AML blasts represents a specific prognostic marker for non-CBF AML with an extended cohort (n=150). We then confirmed our data using a publicly available dataset (BeatAML cohort). This association was also verified within NPM1 mutated patients, which was not associated to co- occurring FLT3-ITD mutations. When further investigating the underlying mechanisms related to CD81 overexpression and its impact on AML progression, we found out that CD81 expression was increased at relapse using paired diagnostic-relapsed samples (19% vs. 67%, P = 0.002). We demonstrated presence of CD81 within the LSC population, defined as CD34+/CD38-/CD90-/CD123+ blasts. Moreover, we showed that the CD81+ LSC fraction was higher at relapse compared to diagnosis (63% vs. 33%, P = 0.0001). In our cohort, non-CBF AML patients with higher level of CD81+LSC had worse outcome (RFS: P = 0.001, HR [95%CI] = 2.01 [1.16-3.48]; OS: P = 0.003; HR [95% CI] = 2.38 [1.32-4.29]. Next, we aimed to explore the potential impact of CD81 expression on LSC function, particularly focusing on its ability to serially engraft in immunocompromised mice (NSG). We observed a positive association between CD81 expression and an increased probability of positive engraftment, and a correlation with time-to-engraftment i.e., faster engraftment of xenografts from AML with high CD81 expression. Ex-vivo drug testing revealed primary AML cells as being more resistant to chemotherapy when engraftment was achieved. To investigate the mechanisms implied, we engineered either overexpression or knocked-down expression of CD81 in three different AML cell lines using cDNA transfection or shRNA, respectively. By means of these controlled CD81 expression models, we were able to demonstrate that CD81 overexpression increased AML cell adhesion, migration and invasion. Imaging by confocal microscopy exposed the development of membrane protrusions for CD81+ models. In vivo, higher CD81 expression increased blast homing, tumor burden and xenoengraftment efficiency. Moreover, we demonstrated that higher CD81 expression was linked to chemoresistance to anthracyclines and cytarabine, the two cornerstone drugs commonly used in AML. Finally, preclinical use of CD81 blocking antibody combined with chemotherapy in xenografts (AML cell line and patient-derived xenografts) resulted in longer relapse-free survival and reduced tumor aggressiveness. These results highlight the functional significance of CD81 overexpression in AML and its potential role as a biomarker for assessing LSC activity. Further investigations may improve this new avenue for targeted therapeutic intervention aimed at disrupting LSC function and improving treatment outcomes in AML patients.

A Dual Role of SOX11 Expression in the Formation of T-Cell Acute Lymphoblastic Leukemia in Mouse Models

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that accounts for 10%-15% of pediatric and 25% of adult ALL cases. Intensified chemotherapy and bone marrow transplants have improved the survival of T-ALL patients. However, around 20% of patients still suffer from primary resistant or relapsed T-ALL. Therefore, improving our knowledge of T-ALL biology and developing precision oncology therapeutics is essential for further progress in T-ALL treatment. SRY-related HMG-box 11 (SOX11) is a transcription factor that regulates the development and survival of multiple embryonic tissues and is crucial for the nervous system. SOX11 is expressed in a multitude of tumor types, where it has been associated with either increased or decreased cell proliferation, metastatic potential, and with either good or bad prognosis, depending on cancer type. Despite being absent in the T-cell lineage, SOX11 is expressed in a subset of T-ALLs, where it plays an unknown role. Based on having a malignantly restricted expression, we hypothesize that SOX11 might be oncogenic in T-ALL. Aim: Identify the role of aberrant SOX11 expression in (pre)leukemic T-cells. Methods: Using a conditional Rosa26-SOX11 overexpression mouse model, we generated mice with T-cell specific (using Lck-cre) SOX11 expression (SOX11 Lck). To assess the self-renewal potential of SOX11 Lck thymocytes, we performed thymic transplantations on sublethally irradiated mice expressing a different allele of CD45. To study SOX11 in a leukemic context, we crossed the SOX11 Lck mice with two established T-ALL mouse models that are either driven by the overexpression of Lmo2 (SOX11 Lck-Lmo2 CD2) or by the loss of the tumor suppressor Pten (SOX11/Pten Lck). An aging cohort of these T-ALL models was followed, and tumor samples were collected. Thymi of 8-weeks old mice were used to study the impact of SOX11 in a pre-leukemic context, and T-cell tumors arising in these models were also used for RNA-sequencing. Using a retroviral strategy, we overexpressed SOX11 in human cord blood stem cells, which we differentiated towards the T-cell lineage ex vivo by co-culturing them on OP9 stromal cells expressing Delta-like Notch ligand 4. Furthermore, we transduced Jurkat cells with a SOX11 expression vector, or with an empty vector, and injected the cells into immunocompromised mice to assess the effect of SOX11 on tumor growth in vivo. Results: In SOX11 Lck mice, we found a significant increase in the immature double-negative 3 (DN3) thymic fraction, with a concomitant decrease in the amount of more mature double-positive CD4 +CD8 + (DP) thymocytes (Fig. A). A possible explanation for this phenotype would be the establishment of a stem-like self-renewal program similar to the one driven by Lmo2. However, by performing thymic transplantations into sublethally irradiated mice, we concluded that SOX11 does not confer self-renewal properties to thymocytes. Additionally, we aged a cohort of SOX11 Lck mice and did not observe formation of T-cell malignancies up to 500 days of age. Given that the expression of SOX11 does not suffice to produce T-cell tumors, we generated the SOX11 Lck-Lmo2 CD2 and SOX11/Pten Lck T-ALL mouse models. In SOX11 Lck-Lmo2 CD2 mice, we found a synergistic effect of the co-expression of both genes on the expansion of DN3 thymocytes (Fig. A), which led to accelerated leukemia formation (Fig. B). By sequencing the RNA of pre-leukemic and leukemic DN3 cells, we observed that SOX11 is mostly regulating cell adhesion, morphology, and cell-cell interactions to promote this oncogenic phenotype in Lmo2-driven T-ALL. On the other hand, SOX11/Pten Lck mice suffered from delayed leukemogenesis (Fig. B), and this tumor suppressor role might be associated with the decrease in DP cells observed in the pre-leukemic stage. Lastly, we set out to validate these findings in a human context. The ex vivo differentiation of human hematopoietic stem cells toward the T-cell lineage showed that SOX11 strongly accelerates early commitment to double-negative T-cell precursors. Also, we observed that Jurkat T-ALL cells engraft faster in immunocompromised mice when expressing SOX11. Conclusions: By generating multiple models of SOX11 expression in the T-cell lineage, we showed that SOX11 impacts T-cell differentiation, both in mice and humans, and that it can promote or hinder T-ALL formation depending on the oncogenic driver.

CLL-1-Targeted Allogeneic CAR-T Cells Exhibit High on-Target Specificity and Potent Cytotoxicity in Preclinical Models of Acute Myeloid Leukemia

Background: CLL-1 is a compelling therapeutic target for acute myeloid leukemia (AML) as it is highly expressed on AML tumor cells and leukemic stem cells, but is not expressed on hematopoietic stem cells. An allogeneic anti-CLL-1 CAR-T cell therapy (CB-012) is in development for relapsed or refractory (r/r) AML. The CAR was generated with a fully human scFv targeting CLL-1 that was selected from a panel of scFvs. CB-012 was engineered with a next-generation Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology to leverage both checkpoint disruption and immune cloaking for potentially improved antitumor activity. Methods: Cas12a chRDNA guides were implemented to generate five genome edits in the manufacture of CB-012. A fully human anti-CLL-1 CAR transgene was site-specifically inserted into the TRAC gene, thereby eliminating TCR expression to reduce graft-versus-host disease. A B2M-HLA-E fusion transgene was inserted into the native B2M gene, preventing expression of all HLA class I antigens except HLA-E, to blunt both T and NK cell-mediated allograft rejection of the CAR-T cells. A knockout of the PDCD1 gene prevented PD-1 receptor expression and thus PD-L1 ligand binding to prolong antitumor activity. This multiplex genome-editing strategy was designed to enhance the antitumor activity of CB-012. In vitro and in vivo studies evaluated specificity for antigen binding, antigen-dependent activity, and preclinical safety assessments. Results: CB-012 CAR-T cells express a fully human anti-CLL-1 scFv-containing CAR construct and demonstrate potent antigen-dependent cytotoxic activity in human AML cell line co-cultures. In AML xenograft models, a single-dose administration of CB-012 CAR-T cells exerted robust tumor control, leading to significant prolongation of survival. Cell binding studies suggested that the anti-CLL-1 scFv does not exhibit tissue cross-reactivity when examined in the context of a collection of cell types representing vital tissues. In an unbiased cell surface protein microarray, the anti-CLL-1 scFv demonstrated specific interaction with human CLL-1, without detectable non-specific interactions. CB-012 CAR-T cells exhibited limited tissue infiltration and expansion in treatment naïve, immunocompromised mouse models. Conclusion: CB-012 demonstrated highly specific and potent CLL-1-targeted cytolytic activity in vitro and in vivo. Specificity of the anti-CLL-1 scFv was further demonstrated by screening in an unbiased protein-binding study and no adverse safety signals were observed in murine models. These data support advancing the development of CB-012 into a first-in-human clinical trial for patients with r/r AML.

Non-Genotoxic Conditioning for Hematopoietic Stem Cell Transplant through Engineered Stem Cell Antibody Paired Evasion (ESCAPE)

While ex vivo gene editing of CD34 + hematopoietic stem and progenitor cells (HSPCs) offers potentially curative therapies for patients with serious diseases, such as sickle cell anemia, significant autologous hematopoietic stem cell (HSC) transplant-related challenges remain, notably the requirement of myeloablative conditioning using chemotherapeutic agents such as busulfan. To address this challenge, we have developed a non-genotoxic conditioning strategy with our Engineered Stem Cell Antibody Paired Evasion (ESCAPE) approach where we aim to engineer the epitope of the HSC surface protein CD117 (cKIT) using base editors, in such a way that the engineered protein retains the normal CD117 receptor function, but escapes recognition by a cognate monoclonal antibody that recognizes the wildtype CD117 protein. The ESCAPE-CD117 edit can be combined with a therapeutic target edit to generate a multiplex-edited engineered HSC (eHSC). We previously reported that through large parallel antibody and antigen screening efforts, we could identify mAb: edit pairs in which the engineered epitope evades mAb-mediated blocking of the WT CD117 receptor-ligand interaction while maintaining normal CD117 receptor biology and HSPC function and differentiation potential in vitro and in vivo. To further characterize binding properties of our lead CD117 variant to the natural ligand SCF, we determined the 3-dimensional structure of the receptor-ligand complex using cryo-EM. Structural overlay between engineered CD117:SCF and WT CD117: SCF complexes showed high similarity, with root mean square deviation (RMSD) of 0.848-Å, indicating that our epitope engineering did not affect the structure of CD117 nor its binding to its ligand. We have previously demonstrated in vivo proof-of-concept data supporting selection of engrafted multiplex-edited eHSCs after a single dose of mAb in non-competitive and competitive transplant settings in multiple rodent xenotransplantation models. We next sought to examine whether mAb conditioning could achieve long-term, multilineage hematopoietic reconstitution of multiplex-edited eHSCs and potentially therapeutic mixed chimerism levels. To this end, we humanized an immunocompromised mouse model with unedited mobilized peripheral blood (mPB) hCD34 + cells to achieve stable human bone marrow chimerism and then administered a single dose of mAb followed by a second transplant from the same donor of eHSCs harboring both ESCAPE and therapeutic edits targeting the HBG1/2 promoter. Animals receiving CD117 mAb demonstrated significant engraftment by cells harboring our ESCAPE multiplex edits as detected by NGS of bulk BM (~50% multiplex editing) and within the sorted marrow CD34 + cell population (~60% multiplex editing), while the isotype control treated animals only showed 2-5% multiplex editing within the said compartments. We also isolated CD15 + myeloid cells and GlyA + erythroid cells from the bone marrow of the mAb treated animals, which also harbored >50% multiplex editing as detected by NGS. Furthermore, we could detect what is believed to be therapeutically relevant levels of HbF induction (>30%) in human GlyA + sorted bone marrow cells, suggesting that we could achieve therapeutic benefit with a single dose of mAb as the conditioning agent. In conclusion, our structural data showed a high level of structural similarity between our engineered CD117 epitope and the WT protein. Our in vitro assays and in vivo rodent xenotransplantation models showed enrichment of engineered HSCs upon mAb treatment. Altogether, our data suggest the feasibility of achieving therapeutically relevant levels of mixed bone marrow chimerism with our non-genotoxic conditioning ESCAPE platform. This approach may remove the need for patients to undergo systemic genotoxic conditioning prior to transplant and further unlock the promise of base editing therapies for the treatment of patients suffering from serious hematologic diseases.

Antitumor Immunity Mediated by Photodynamic Therapy Using Injectable Chitosan Hydrogels for Intratumoral and Sustained Drug Delivery.

Photodynamic therapy (PDT) is an anticancer therapy with proven efficacy; however, its application is often limited by prolonged skin photosensitivity and solubility issues associated with the phototherapeutic agents. Injectable hydrogels which can effectively provide intratumoral delivery of photosensitizers with sustained release are attracting increased interest for photodynamic cancer therapies. However, most of the hydrogels for PDT applications are based on systems with high complexity, and often, preclinical validation is not provided. Herein, we provide a simple and reliable pH-sensitive hydrogel formulation that presents appropriate rheological properties for intratumoral injection. For this, Temoporfin (m-THPC), which is one of the most potent clinical photosensitizers, was chemically modified to introduce functional groups that act as cross-linkers in the formation of chitosan-based hydrogels. The introduction of -COOH groups resulted in a water-soluble derivative, named PS2, that was the most promising candidate. Although PS2 was not internalized by the target cells, its extracellular activation caused effective damage to the cancer cells, which was likely mediated by lipid peroxidation. The injection of the hydrogel containing PS2 in the tumors was monitored by high-frequency ultrasounds and in vivo fluorescence imaging which confirmed the sustained release of PS2 for at least 72 h. Following local administration, light exposure was conducted one (single irradiation protocol) or three (multiple irradiation protocols) times. The latter delivered the best therapeutic outcomes, which included complete tumor regression and systemic anticancer immune responses. Immunological memory was induced as ∼75% of the mice cured with our strategy rejected a second rechallenge with live cancer cells. Additionally, the failure of PDT to treat immunocompromised mice bearing tumors reinforces the relevance of the host immune system. Finally, our strategy promotes anticancer immune responses that lead to the abscopal protection against distant metastases.

Introduction of Androgen Receptor Targeting shRNA Inhibits Tumor Growth in Patient-Derived Prostate Cancer Xenografts.

Patient-derived xenograft (PDX) models have been established as important preclinical cancer models, overcoming some of the limitations associated with the use of cancer cell lines. The utility of prostate cancer PDX models has been limited by an inability to genetically manipulate them in vivo and difficulties sustaining PDX-derived cancer cells in culture. Viable, short-term propagation of PDX models would allow in vitro transfection with traceable reporters or manipulation of gene expression relevant to different studies within the prostate cancer field. Here, we report an organoid culture system that supports the growth of prostate cancer PDX cells in vitro and permits genetic manipulation, substantially increasing the scope to use PDXs to study the pathobiology of prostate cancer and define potential therapeutic targets. We have established a short-term PDX-derived in vitro cell culture system which enables genetic manipulation of prostate cancer PDXs LuCaP35 and BM18. Genetically manipulated cells could be re-established as viable xenografts when re-implanted subcutaneously in immunocompromised mice and were able to be serially passaged. Tumor growth of the androgen-dependent LuCaP35 PDX was significantly inhibited following depletion of the androgen receptor (AR) in vivo. Taken together, this system provides a method to generate novel preclinical models to assess the impact of controlled genetic perturbations and allows for targeting specific genes of interest in the complex biological setting of solid tumors.

Evaluation of the safety and efficiency of cytotoxic T cell therapy sensitized by tumor antigens original from T-ALL-iPSC in vivo.

Since RNA sequencing has shown that induced pluripotent stem cells (iPSCs) share a common antigen profile with tumor cells, cancer vaccines that focus on iPSCs have made promising progress in recent years. Previously, we showed that iPSCs derived from leukemic cells of patients with primary T cell acute lymphoblastic leukemia (T-ALL) have a gene expression profile similar to that of T-ALL cell lines. Mice with T-ALL were treated with dendritic and T (DC-T) cells loaded with intact and complete antigens from T-ALL-derived iPSCs (T-ALL-iPSCs). We evaluated the safety and antitumor efficiency of autologous tumor-derived iPSC antigens by flow cytometry, cytokine release assay, acute toxicity experiments, long-term toxicity experiments, and other methods. Our results indicate that complete tumor antigens from T-ALL-iPSCs could inhibit the growth of inoculated tumors in immunocompromised mice without causing acute and long-term toxicity. T-ALL-iPSC-based treatment is safe and can be used as a potential strategy for leukemia immunotherapy.

Regulatory Effects of Ganoderma lucidum, Grifola frondosa, and American ginseng Extract Formulation on Gut Microbiota and Fecal Metabolomics in Mice.

The bioactivities of Ganoderma lucidum, Grifola frondosa, and American ginseng have been extensively studied and documented. However, the effects of their complexes on the structural properties of intestinal microbiota and fecal metabolism remain unclear. Therefore, this paper aims to present a preliminary study to shed light on this aspect. In this study, an immunocompromised mouse model was induced using cyclophosphamide, and Ganoderma lucidum, Grifola frondosa, and American ginseng extract formulation (referred to as JGGA) were administered via gavage to investigate their modulatory effects on gut microbiota and fecal metabolism in mice. The effects of JGGA on immune enhancement were explored using serum test kits, hematoxylin-eosin staining, 16SrDNA high-throughput sequencing, and UHPLC-QE-MS metabolomics. The findings revealed potential mechanisms underlying the immune-enhancing effects of JGGA. Specifically, JGGA administration resulted in an improved body weight, thymic index, splenic index, carbon scavenging ability, hypersensitivity, and cellular inflammatory factor expression levels in mice. Further analysis demonstrated that JGGA reduced the abundance of Firmicutes, Proteobacteria, and Actinobacteria, while increasing the abundance of Bacteroidetes. Additionally, JGGA modulated the levels of 30 fecal metabolites. These results suggest that the immune enhancement observed with JGGA may be attributed to the targeted modulation of gut microbiota and fecal metabolism, thus promoting increased immunity in the body.