Human CD34 Research Articles

CD47;Rag2;IL-2rγ triple knock-out mice pre-conditioning with busulfan could be a novel platform for generating hematopoietic stem cells engrafted humanized mice.

Humanized mouse models to recapitulate human biological systems still have limitations, such as the onset of lethal graft-versus-host disease (GvHD), a variable success rate, and the low accessibility of total body irradiation (TBI). Recently, mice modified with the CD47-SIRPA axis have been studied to improve humanized mouse models. However, such trials have been rarely applied in NOD mice. In this study, we created a novel mouse strain, NOD-CD47nullRag2nullIL-2rγnull (RTKO) mice, and applied it to generate humanized mice. Four-week-old female NOD-Rag2nullIL-2rγnull (RID) and RTKO mice pre-conditioned with TBI or busulfan (BSF) injection were used for generating human CD34+ hematopoietic stem cell (HSC) engrafted humanized mice. Clinical signs were observed twice a week, and body weight was measured once a week. Flow cytometry for human leukocyte antigens was performed at intervals of four weeks or two weeks, and mice were sacrificed at 48 weeks after HSC injection. For a long period from 16 to 40 weeks post transplantation, the percentage of hCD45 was mostly maintained above 25% in all groups, and it was sustained the longest and highest in the RTKO BSF group. Reconstruction of human leukocytes, including hCD3, was also most prominent in the RTKO BSF group. Only two mice died before 40 weeks post transplantation in all groups, and there were no life-threatening GvHD lesions except in the dead mice. The occurrence of GvHD has been identified as mainly due to human T cells infiltrating tissues and their related cytokines. Humanized mouse models under all conditions applied in this study are considered suitable models for long-term experiments based on the improvement of human leukocytes reconstruction and the stable animal health. Especially, RTKO mice pretreated with BSF are expected to be a valuable platform not only for generating humanized mice but also for various immune research fields.

Rectifying the impairment of immune thrombocytopenia plasmas through photobiomodulation

<p>Immune thrombocytopenia (ITP) is an autoimmune hemorrhage disorder. The first-line treatment of this disorder is corticosteroids, followed by thrombopoietin (TPO) receptor agonists such as Nplate, and/or splenectomy. Yet, the extended usage of corticosteroids or the expensive Nplate, coupled with the implications of splenectomy, raises concerns due to the array of associated side effects and an escalated vulnerability to subsequent complications. The current investigation shows that while anti-platelet antibodies and ITP plasmas hinder megakaryocyte differentiation and maturation and impair proplatelet and platelet formation in ex vivo culture of umbilical cord human CD34+ stem cells (cHSCs), low-level laser (LLL) treatment or photobiomodulation (PBM) effectively mitigates these detrimental impacts. PBM reinstated megakaryocyte differentiation and maturation, bolstering proplatelet and platelet formation in the presence of auto-platelet antibodies or ITP plasmas. The mitigating effects of PBM appear to pivot on its capacity to uphold cellular mitochondrial functionality and rectify the mitochondrial impairments engendered by anti-platelet antibodies or ITP plasmas. These findings underscore the potential of PBM as a safe and cost-efficient alternative for the management of a specific subset of ITP patients.</p>

Rectifying the impairment of immune thrombocytopenia plasmas through photobiomodulation

<p>Immune thrombocytopenia (ITP) is an autoimmune hemorrhage disorder. The first-line treatment of this disorder is corticosteroids, followed by thrombopoietin (TPO) receptor agonists such as Nplate, and/or splenectomy. Yet, the extended usage of corticosteroids or the expensive Nplate, coupled with the implications of splenectomy, raises concerns due to the array of associated side effects and an escalated vulnerability to subsequent complications. The current investigation shows that while anti-platelet antibodies and ITP plasmas hinder megakaryocyte differentiation and maturation and impair proplatelet and platelet formation in ex vivo culture of umbilical cord human CD34+ stem cells (cHSCs), low-level laser (LLL) treatment or photobiomodulation (PBM) effectively mitigates these detrimental impacts. PBM reinstated megakaryocyte differentiation and maturation, bolstering proplatelet and platelet formation in the presence of auto-platelet antibodies or ITP plasmas. The mitigating effects of PBM appear to pivot on its capacity to uphold cellular mitochondrial functionality and rectify the mitochondrial impairments engendered by anti-platelet antibodies or ITP plasmas. These findings underscore the potential of PBM as a safe and cost-efficient alternative for the management of a specific subset of ITP patients.</p>

Deciphering Acute Myeloid Leukemia Associated Transcription Factors in Human Primary CD34+ Hematopoietic Stem/Progenitor Cells.

Hemato-oncological diseases account for nearly 10% of all malignancies and can be classified into leukemia, lymphoma, myeloproliferative diseases, and myelodysplastic syndromes. The causes and prognosis of these disease entities are highly variable. Most entities are not permanently controllable and ultimately lead to the patient's death. At the molecular level, recurrent mutations including chromosomal translocations initiate the transformation from normal stem-/progenitor cells into malignant blasts finally floating the patient's bone marrow and blood system. In acute myeloid leukemia (AML), the so-called master transcription factors such as RUNX1, KMT2A, and HOX are frequently disrupted by chromosomal translocations, resulting in neomorphic oncogenic fusion genes. Triggering ex vivo expansion of primary human CD34+ stem/progenitor cells represents a distinct characteristic of such chimeric AML transcription factors. Regarding oncogenic mechanisms of AML, most studies focus on murine models. However, due to biological differences between mice and humans, findings are only partly transferable. This review focuses on the genetic manipulation of human CD34+ primary hematopoietic stem/progenitor cells derived from healthy donors to model acute myeloid leukemia cell growth. Analysis of defined single- or multi-hit human cellular AML models will elucidate molecular mechanisms of the development, maintenance, and potential molecular intervention strategies to counteract malignant human AML blast cell growth.

Generation of human ILC3 from allogeneic and autologous CD34+ hematopoietic progenitors toward adoptive transfer

Type 3 innate lymphoid cells (ILC3) are important in tissue homeostasis. In the gut, ILC3 repair damaged epithelium and suppress inflammation. In allogeneic hematopoietic cell transplantation (HCT), ILC3 protect against graft-versus-host disease (GvHD), most likely by restoring tissue damage and preventing inflammation. We hypothesize that supplementing HCT grafts with interleukin-22 (IL-22)-producing ILC3 may prevent acute GvHD. We therefore explored ex vivo generation of human IL-22-producing ILC3 from hematopoietic stem and progenitor cells (HSPC) obtained from adult, neonatal and fetal sources. We established a stroma-free system culturing human cord blood-derived CD34+ HSPC with successive cytokine mixes for 5 weeks. We analyzed the presence of phenotypically defined ILC, their viability, proliferation and IL-22 production (after stimulation) by flow cytometry and enzyme-linked immunosorbent assay (ELISA). We found that the addition of recombinant human IL-15 and the enhancer of zeste homolog 1/2 inhibitor UNC1999 promoted ILC3 generation. Similar results were demonstrated when UNC1999 was added to CD34+ HSPC derived from healthy adult granulocyte colony-stimulating factor mobilized peripheral blood and bone marrow, but not fetal liver. UNC1999 did not negatively impact IL-22 production in any of the HSPC sources. Finally, we observed that autologous HSPC mobilized from the blood of adults with hematological malignancies also developed into ILC3, albeit with a significantly lower capacity. Together, we developed a stroma-free protocol to generate large quantities of IL-22-producing ILC3 from healthy adult human HSPC that can be applied for adoptive transfer to prevent GvHD after allogeneic HCT.

In vitro differentiation of human CD45+ CD34+ induced hematopoietic progenitor cells from iPSCs using a monolayer system

Hematopoietic stem cells (HSCs) represent crucial target cells in the management of hematopoietic and immune system disorders. Unfortunately, the primary source of hematopoietic stem cells is limited. Hematopoietic stem cells derived from induced pluripotent stem cells (iPSCs) hold great promise for applications in cell therapy, disease modeling, and drug screening. To achieve a consistent induction method, one specific induction scheme capable of reliably generating CD34 and CD45 double-positive cells from iPSCs was optimized, employing a comparative analysis and screening of various induction methods. The comprehensive induction procedures are outlined in this document.

Human CD34+/dim neutrophil-committed progenitors do not differentiate into neutrophil-like CXCR1+CD14+CD16- monocytes in vitro.

The advent of recent cutting-edge technologies has allowed the discovery and characterization of novel progenitors of human neutrophils, including SSCloCD66b+CD15+CD11b-CD49dhiproNeu1s, SSChiCD66b+CD15+CD11b-CD49dintproNeus2s, CD66b+CD15+CD11b+CD49d+CD101-preNeus, and Lin-CD66b+CD117+CD71+eNePs. In this research field, we recently identified CD66b-CD38+CD64dimCD115-, CD34+, and CD34dim/- cells exclusively committed to the neutrophil lineage (which we renamed as CD34+ and CD34dim/- neutrophil-committed progenitors), representing the earliest neutrophil precursors identifiable and sorted by flow cytometry. Moreover, based on their differential CD34 and CD45RA expression, we could identify 4 populations of neutrophil-committed progenitors: CD34+CD45RA-/NCP1s, CD34+CD45RA+/NCP2s, CD34dim/-CD45RA+/NCP3s, and CD34dim/-CD45RA-/NCP4s. This said, a very recent study by Ikeda and coworkers (PMID: 36862552) reported that neutrophil precursors, termed either neutrophil progenitors or "early neutrophil-committed progenitors," would generate immunosuppressive neutrophil-like CXCR1+CD14+CD16- monocytes. Hence, presuming that neutrophil progenitors/"early neutrophil-committed progenitors" correspond to neutrophil-committed progenitors, the selective neutrophil commitment that we attributed to neutrophil-committed progenitors is contradicted by Ikeda and coworkers' article. In this study, by performing a more analytical reevaluation at the phenotypic and molecular levels of the cells generated by neutrophil-committed progenitors 2 and 4 (selected as representatives of neutrophil-committed progenitors), we categorically exclude that neutrophil-committed progenitors generate neutrophil-like CXCR1+CD14+CD16- monocytes. Rather, we provide substantial evidence indicating that the cells generated by neutrophil progenitors/"early neutrophil-committed progenitors" are neutrophilic cells at a different stage of maturation, displaying moderate levels of CD14, instead of neutrophil-like CXCR1+CD14+CD16- monocytes, as pointed by Ikeda and coworkers. Hence, the conclusion that neutrophil progenitors/"early neutrophil-committed progenitors" aberrantly differentiate into neutrophil-like monocytes derives, in our opinion, from data misinterpretation.

MarrowCellDLD: a microfluidic method for label-free retrieval of fragile bone marrow-derived cells

Functional bone marrow studies have focused primarily on hematopoietic progenitors, leaving limited knowledge about other fragile populations, such as bone marrow adipocytes (BMAds) and megakaryocytes. The isolation of these cells is challenging due to rupture susceptibility and large size. We introduce here a label-free cytometry microsystem, MarrowCellDLD, based on deterministic lateral displacement. MarrowCellDLD enables the isolation of large, fragile BM-derived cells based on intrinsic size properties while preserving their viability and functionality. Bone marrow adipocytes, obtained from mouse and human stromal line differentiation, as well as megakaryocytes, from primary human CD34+ hematopoietic stem and progenitor cells, were used for validation. Precise micrometer-range separation cutoffs were adapted for each cell type. Cells were sorted directly in culture media, without pre-labeling steps, and with real-time imaging for quality control. At least 106 cells were retrieved intact per sorting round. Our method outperformed two FACS instruments in purity and yield, particularly for large cell size fractions. MarrowCellDLD represents a non-destructive sorting tool for large, fragile BM-derived cells, facilitating the separation of pure populations of BMAds and megakaryocytes to further investigate their physiological and pathological roles.

Preclinical evaluation of the efficacy of an antibody to human SIRPα for cancer immunotherapy in humanized mouse models.

Tumor-associated macrophages (TAMs) are abundant in the tumor microenvironment and are considered potential targets for cancer immunotherapy. To examine the antitumor effects of agents targeting human TAMs in vivo, we here established preclinical tumor xenograft models based on immunodeficient mice that express multiple human cytokines and have been reconstituted with a human immune system by transplantation of human CD34+ hematopoietic stem and progenitor cells (HIS-MITRG mice). HIS-MITRG mice supported the growth of both human cell line (Raji)- and patient-derived B cell lymphoma as well as the infiltration of human macrophages into their tumors. We examined the potential antitumor action of an antibody to human SIRPα (SE12C3) that inhibits the interaction of CD47 on tumor cells with SIRPα on human macrophages and thereby promotes Fcγ receptor-mediated phagocytosis of the former cells by the latter. Treatment with the combination of rituximab (antibody to human CD20) and SE12C3 inhibited Raji tumor growth in HIS-MITRG mice to a markedly greater extent than did rituximab monotherapy. This enhanced antitumor effect was dependent on human macrophages and attributable to enhanced rituximab-dependent phagocytosis of lymphoma cells by human macrophages. Treatment with rituximab and SE12C3 also induced reprogramming of human TAMs toward a proinflammatory phenotype. Furthermore, the combination treatment essentially prevented the growth of patient-derived diffuse large B cell lymphoma in HIS-MITRG mice. Our findings thus support the study of HIS-MITRG mice as a model for the preclinical evaluation in vivo of potential therapeutics, such as antibodies to human SIRPα, that target human TAMs.

Deficiency of thioredoxin-interacting protein results in age-related thrombocytopenia due to megakaryocyte oxidative stress

BackgroundPlatelets are generated from megakaryocytes (MKs), mainly located in the bone marrow (BM). Megakaryopoiesis can be affected by genetic disorders, metabolic diseases, and aging. The molecular mechanisms underlying platelet count regulation have not been fully elucidated. ObjectivesIn the present study, we investigated the role of thioredoxin-interacting protein (TXNIP), a protein that regulates cellular metabolism in megakaryopoiesis, using a Txnip−/− mouse model. MethodsWild-type (WT) and Txnip−/− mice (2-27-month-old) were studied. BM-derived MKs were analyzed to investigate the role of TXNIP in megakaryopoiesis with age. The global transcriptome of BM-derived CD41+ megakaryocyte precursors (MkPs) of WT and Txnip−/− mice were compared. The CD34+ hematopoietic stem cells isolated from human cord blood were differentiated into MKs. ResultsTxnip−/− mice developed thrombocytopenia at 4 to 5 months that worsened with age. During ex vivo megakaryopoiesis, Txnip−/− MkPs remained small, with decreased levels of MK-specific markers. Critically, Txnip−/− MkPs exhibited reduced mitochondrial reactive oxygen species, which was related to AKT activity. Txnip−/− MkPs also showed elevated glycolysis alongside increased glucose uptake for ATP production. Total RNA sequencing revealed enrichment for oxidative stress- and apoptosis-related genes in differentially expressed genes between Txnip−/− and WT MkPs. The effects of TXNIP on MKs were recapitulated during the differentiation of human cord blood-derived CD34+ hematopoietic stem cells. ConclusionWe provide evidence that the megakaryopoiesis pathway becomes exhausted with age in Txnip−/− mice with a decrease in terminal, mature MKs that response to thrombocytopenic challenge. Overall, this study demonstrates the role of TXNIP in megakaryopoiesis, regulating mitochondrial metabolism.

Characterization of Human Engraftment and Hemophagocytic Lymphohistiocytosis in NSG-SGM3 Neonate Mice Engrafted with Purified CD34+ Hematopoietic Stem Cells

Immunodeficient mice bearing human immune systems, or "humanized" chimeric mice, are widely used in basic research, along with the preclinical stages of drug development. Nonobese diabetic-severe combined immunodeficiency (NOD-SCID) IL2Rγnull (NSG) mice expressing human stem cell factor, granulocyte-macrophage colony stimulating factor, and interleukin-3 (NSG-SGM3) support robust development of human myeloid cells and T cells but have reduced longevity due to the development of fatal hemophagocytic lymphohistiocytosis (HLH). Here, we describe an optimized protocol for development of human immune chimerism in NSG-SGM3 mice. We demonstrate that efficient human CD45+ reconstitution can be achieved and HLH delayed by engraftment of neonatal NSG-SGM3 with low numbers of human umbilical cord-derived CD34+ hematopoietic stem cells in the absence of preconditioning irradiation.

Development of Physiologically Relevant Skin Organoids from Human Induced Pluripotent Stem Cells.

The development of skin organs for studying developmental pathways, modeling diseases, or regenerative medicine purposes is a major endeavor in the field. Human induced pluripotent stem cells (hiPSCs) are successfully used to deriveskin cells, but the field is still far from meeting the goal of creating skin containing appendages, such as hair follicles and sweat glands. Here, the goal is to generate skin organoids (SKOs) from human skin fibroblast or placental CD34+ cell-derived hiPSCs. With all three hiPSC lines, complex SKOs with stratified skin layers and pigmented hair follicles are generated with different efficacies. In addition, the hiPSC-derived SKOs develop sebaceous glands, touch-receptive Merkel cells, and more importantly eccrine sweat glands. Together, physiologically relevant skin organoids are developed by direct induction of embryoid body formation, along with simultaneous inactivation of transforming growth factor beta signaling, activation of fibroblast growth factor signaling, and inhibition of bone morphogenetic protein signaling pathways. The skin organoids created in this study can be used as valuable platforms for further research into human skin development, disease modeling, or reconstructive surgeries.

Kidney Immune Cell Characterization of Humanized Mouse Models.

Experimental studies often fail to translate to clinical practice. Humanized mouse models are an important tool to close this gap. We immunophenotyped the kidneys of NOG (EXL) and NSG mouse strains engrafted with human CD34 + hematopoietic stem cells or PBMCs and compared with immune cell composition of normal human kidney. Human CD34 + hematopoietic stem cell engraftment results in steady renal immune cell populations in mouse kidney with key similarities in composition compared with human kidney. Successful translation of experimental mouse data to human diseases is limited because of biological differences and imperfect disease models. Humanized mouse models are being used to bring murine models closer to humans. However, data for application in renal immune cell-mediated diseases are rare. We therefore studied immune cell composition of three different humanized mouse kidneys and compared them with human kidney. NOG and NOGEXL mice engrafted with human CD34 + hematopoietic stem cells were compared with NSG mice engrafted with human PBMCs. Engraftment was confirmed with flow cytometry, and immune cell composition in kidney, blood, spleen, and bone marrow was analyzed in different models. The results from immunophenotyping of kidneys from different humanized mouse strains were compared with normal portions of human kidneys. We found significant engraftment of human immune cells in blood and kidney of all tested models. huNSG mice showed highest frequencies of hTCR + cells compared with huNOG and huNOGEXL in blood. huNOGEXL was found to have the highest hCD4 + frequency among all tested models. Non-T cells such as hCD20 + and hCD11c + cells were decreased in huNSG mice compared with huNOG and huNOGEXL. Compared with normal human kidney, huNOG and huNOGEXL mice showed representative immune cell composition, rather than huNSG mice. In summary, humanization results in immune cell infiltration in the kidney with variable immune cell composition of tested humanized mouse models and partially reflects normal human kidneys, suggesting potential use for translational studies.

Abstract A016: Novel LEF1 gene structural variants in T-cell acute lymphoblastic leukemia Patient-Derived Xenografts models

Abstract Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is a malignant hematological disorder characterized by uncontrolled proliferation of immature T-cell precursors. LEF1 (Lymphoid Enhancer-Binding Factor 1) is a transcription factor that plays a crucial role in the development of T-cells. Recent studies have shown that LEF1 can affect the expression of MYC, a proto-oncogene that is frequently dysregulated in cancer. The highly expressed LEF1 gene and potentially its variants (K86E and P106L) stimulate cell growth by upregulating the expression of MYC. In T-ALL, MYC is often overexpressed, the abnormal expression of MYC propels the advancement of T-ALL and sustains the function of leukemia stem cells. The interaction between LEF1 and MYC in T-ALL is complex and involves multiple regulatory pathways, particularly the Wnt/β-catenin signaling pathway. Several studies have revealed a significant correlation between the inactivation of LEF1 and the overexpression of MYC in T-ALL. Therefore, understanding the role of LEF1 in T-ALL pathogenesis may provide new insights into the development of novel therapeutic strategies for this aggressive disease. This study shows that the novel structural variants (SVs) found in the LEF1 gene could be associated with the decreased expression of MYC. Methods: Leukemia cells derived from 6 patients with T-ALL were inoculated intravenously into immunodeficient mice tails. The burden of tumor was monitored within the peripheral blood (hCD45+), and the leukemia cells were collected once human CD45 was above 80%. Optical Genome Mapping (OGM, Bionano Genomics) and Whole Genome Sequencing (WGS) techniques were used to detect SVs and mutations. The findings were subsequently validated using polymerase chain reaction (PCR). Results: Of the 6 T-ALL models that were analyzed, two of them had SVs in the LEF1 gene. Although the expression of the LEF1 gene did not show significant changes, the expression of MYC was observed to be downregulated. The OGM analysis revealed that the second and third exons of the LEF1 gene, which are located in the β-catenin binding region, were deleted at the DNA level. The lack of complete identity between the sequences at the breakpoints could be attributed to DNA repair mechanisms. At the RNA level, the two models had distinct LEF1 mutations - E60K and P106L. Conclusion: In two of the six T-ALL models, novel SVs were found in the LEF1 gene that may decrease MYC expression compared to control samples. Further research is required to understand the relationship between LEF1 deletion and the regulation of MYC expression, as well as the role of this phenomenon in the development of T-ALL. Targeting LEF1 has emerged as a potential therapeutic strategy for the treatment of T-ALL. Citation Format: Yueying Wang, Wubin Qian, Mengting Qin, Xiaobo Chen, Jia Xue, Sheng Guo. Novel LEF1 gene structural variants in T-cell acute lymphoblastic leukemia Patient-Derived Xenografts models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A016.

Abstract A024: Systemic administration of CD3-redirected lentiviral vector efficiently targets CD19-CAR delivery to human T cells in vivo

Abstract In recent years, there has been a significant increase in interest regarding the generation of CAR-T cells directly within patients using a simple vector injection containing the CAR transgene. This promising technology has the potential to streamline the procedure and decrease its overall cost. To ensure effective in vivo delivery, these vectors must demonstrate resistance to neutralization by human serum, the ability to specifically target and transduce desired cells, and must support their subsequent expansion. Lentiviral vectors (LV) pseudotyped with VSV G-protein (G-WT) have a broad tropism, targeting various cell types through their affinity for the ubiquitously expressed LDL receptor (LDLR). G-WT LV have proven effective in generating therapeutic CAR-T cells ex vivo, however, requiring proper purification and activation of T cells beforehand. To address challenges related to their in vivo delivery, we utilized rational engineering to redesign the G protein, resulting in a modified version termed G-CD3. This redesigned G protein incorporates a CD3-specific single-chain antibody, allowing for targeted recognition of T cells while avoiding LDLR recognition. Upon testing the newly designed G-CD3 LV, we demonstrated its specificity in targeting and infecting both un-activated and activated T cells in peripheral blood monocytic cell cultures. In addition, the G-CD3 LV powerfully activates T cells, evident from their increased CD25 expression, and the infection is not hindered by the presence of human serum (25%). In further experiments, we successfully demonstrated that G-CD3-LV can infect T cells in fresh whole blood. In vivo evaluation of G-CD3-LV efficiency was performed in humanized NSG mice with more than 40 % of human CD45+ cells by intravenous (IV) or intraperitoneal (IP) LV delivery using LV encoding anti-CD19 CAR and GFP transgenes. Both IV and IP delivery of G-CD3-LV led to in vivo CAR T cell generation. However, CAR-T blood levels were higher after IP administration, reaching 13-17% of total circulating human CD45+ cells at the peak on day 21. Anti CD19-CAR-T cell generation coincided with the disappearance of endogenous CD19+ B cells, demonstrating functional in vivo CAR-T generation. Tissue analysis at day 39 after LV injection showed the presence of anti-CD19 CAR-T cells accompanied by the absence or dramatic reduction in CD19+ B cells in spleen, bone marrow and liver. In contrast, no CAR-T cell generation was detected at any time point in the mice injected with G-WT-LV, regardless of whether the virus was administered IV or IP. Taken together these data highlight the potential applicability of newly designed retargeted LV for in vivo gene delivery, offering promising prospects for future human gene therapy applications. Citation Format: Karina Krotova, Gopal Naik Nenavath, Nandakumar Packiriswamy, Rianna Vandergaast, Christopher Ziegler, Melissa Moy, Riya Narjari, Luke Schnebeck, Zachary Larson, Kyle Gromer, Chia-Hsuan Chin, Samantha Reiter, Miguel Muñoz Alía, Kah-Whye Peng, Stephen Russell. Systemic administration of CD3-redirected lentiviral vector efficiently targets CD19-CAR delivery to human T cells in vivo [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A024.

Abstract A066: TUSC2 immunogene therapy enhances checkpoint blockade through increased cytotoxic immune activation in chemo-resistant small cell lung cancer (SCLC) in humanized mice

Abstract Immunotherapy has been effective but has had limited effect in clinical trials for SCLC. We identified TUSC2, a potent tumor suppressor gene, as a novel immunogene, which mediates both apoptosis and induces a strong antitumor immune response. TUSC2 expression is reduced in almost all SCLC tumors and absent in 41%. We investigated the antitumor immune response to TUSC2 gene therapy in combination with immune checkpoint blockade (ICB) against a chemo-resistant inflamed SCLC tumors in humanized mice. Colony formation was markedly reduced in platinum resistant inflamed H841 cells transfected with TUSC2. Humanized mice were generated by transplanting fresh human cord blood derived CD34 stem cells into sub-lethally irradiated NSG mice(hu-NSG). The level of engraftment of human CD45, CD3 T, CD19 B, NK cells was verified before tumor implantation at > 25%. Subcutaneous tumors were developed in hu-NSG mice and treated with intravenous injections of TUSC2 loaded cationic lipid nanoparticles (quaratusugene ozeplasmid abbreviated quar oze) with or without pembrolizumab (pembro; anti-PD1). A dramatic antitumor effect was mediated by quar oze, whereas pembro remained ineffective. A synergistic antitumor effect was found when quar oze was combined with pembro. The antitumor effect of the quar oze + pembro was associated with increased infiltration of human TILs, CD3 T, cytotoxic T, and NK cells, and a decreased number of human regulatory T (Treg) cells, and PD1 expressing exhausted CD8 T cells in the tumor microenvironment. Quar oze was also combined with atezolizumab (atezo; anti-PD-L1) and the antitumor effect on H841 lung metastases in hu-NSG mice was tested. Metastases were treated with quar oze and atezo alone or in combination. Although quar oze alone and atezo alone showed a strong antitumor effect in controlling metastases, a greater antitumor effect was found when quar oze was combined with atezo. Infiltration of human CD45, CD3 T, CD8 T and NK cells increased in the quar oze and quar oze +atezo treated groups compared with others. A significantly higher number of CD8 T effector memory and lower number of naïve T cells were found in the quar oze +atezo which were associated with increased activated CD8 T cells (CD69+, PD1+ CD8 T cells). HLA-DR+ dendritic cells were increased, whereas myeloid derived suppressor cells were strongly inhibited by the combination. Quar oze was also combined with carboplatin+atezo which showed enhanced antitumor efficacy compared with carbo+atezo or quar oze alone. When quar oze was combined with carboplatin, a greater effect was found in inhibiting H841 cell growth, colony formation, and induction of apoptosis. Quar oze was combined with NPRL2, another tumor suppressor gene, in H841 tumors in humanized mice. Synergistic antitumor efficacy of the dual gene therapy occurred with the quar oze +NPRL2 treatment. Taken together, these data suggest that TUSC2 gene therapy in combination with ICB induces strong antitumor activity on a chemo-resistant inflamed SCLC through cytotoxic CD8 T and NK cell activation. Citation Format: Ismail M Meraz, Mourad Majidi, Renduo Song, Feng Meng, Lihui Gao, Qi Wang, Jing Wang, Elizabeth Shpall, Mark Berger, Hemant Kumar, Jack A Roth. TUSC2 immunogene therapy enhances checkpoint blockade through increased cytotoxic immune activation in chemo-resistant small cell lung cancer (SCLC) in humanized mice [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A066.

SR-18292 Induces Fetal Hemoglobin Synthesis and Reduces Disease Pathology in Sickle Cell Mice

Sickle cell disease (SCD) is a commonly inherited blood disorder, which affects millions around the world. Reactivation of fetal hemoglobin (HbF) synthesis in adult erythroid cells can diminish the severity of many clinical features of the disease. Up-regulation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) has been shown to induce HbF levels in human primary erythroid progenitor CD34 + cells. Here we reported the in vitro and in vivo effects of a small molecular compound, SR-18292, on HbF induction via modulating PGC-1α. To determine whether SR-18292 can induce HbF in human CD34 + cells, we performed RT-qPCR, flow cytometry, and western blot to measure the mRNA and protein levels of PGC-1α and γ-globin. We found that mRNA levels of PGC-1α and γ-globin were significantly increased in cells treated with SR-18292 while β-globin mRNA levels were unaffected. Protein levels of γ-globin and the ratio of F-cells were increased in treated cells. Erythroid cellular differentiation markers (CD71/CD235a) and terminal differentiation markers (CD49d/CD233) were used to examine the differentiation status. There was no significant difference in the population of CD71 +CD235a + cells among all groups. However, SR-18292 treatment increased the percentage of CD49d -CD233 + cells at 5 or 7.5 μM. These results suggest that SR-18292 at higher concentrations can promote erythroid terminal differentiation. To evaluate whether the HbF induction by SR-18292 can reduce the SCD disease pathology in SCD mice or not, we intraperitoneally administered SR-18292 to SCD mice. HbF induction was determined by flow cytometric analysis using an anti-HbF antibody. And it was shown that the HbF-high cell population was induced in SCD mice after 4 weeks of treatment. And we used transferrin receptor CD71 and erythroid-specific marker Ter119 to determine whether SR-18292 could affect erythroid differentiation. There was no significant difference in the CD71 +Ter119 + population after 4 weeks of SR-18192 treatment compared with untreated animals. The mRNA levels of γ-globin and PGC-1α were increased in treated mice. And the increase of PGC-1α and γ-globin protein levels were confirmed by western blot. Reticulocyte is a sign of premature RBC destruction (hemolysis), and the SCD mice usually have high reticulocyte counts (>50%). We measured the reticulocyte in the peripheral blood of SR-18292 treated and untreated SCD mice by flow-cytometric analyses and found that reticulocyte was significantly reduced in SR-18292 treated animals (from 55% to 20%) after 4 weeks of treatment. Complete blood cell counts (CBC) showed that SR-18292 treated mice had slightly elevated numbers of red blood cells (RBCs) and more hemoglobin, but lower counts of white blood cells, lymphocytes, and neutrophils. In addition, the RBC distribution width (RDW) was significantly reduced after SR-18292 treatment, indicating that the size of circulating RBCs was more uniform in treated mice. In addition, erythroid cell morphology was evaluated by blood smears with Wright-Giemsa stain. The number of sickled RBCs were reduced in SCD mice treated with SR-18292 for 4 weeks. This is consistent with our observation that RDW was reduced in SCD mice treated with SR-18292. Furthermore, SR-18292 treated mice had a statistically significant reduction in spleen size and weight in contrast to the spleens of untreated SCD mice. The indices of RBCs were improved in accompany with reduced hemolysis and a higher fraction of circulating mature erythrocytes in treated SCD mice. There were no observable adverse effects on other hematopoietic cell lineages. We also performed single-cell RNA-seqs in human CD34 + cells treated with SR-18292 and found that some known HbF repressors (such as BCL11A and ZBTB7A) were decreased in treated groups. Besides that, there were many differentially expressed genes (DEGs), which might be novel HbF regulators. We are verifying those DEGs genes by qPCR and using CRISPR/cas9 to examine their functions in globin regulation. In summary, our findings suggest that SR-18292 might be a promising therapeutic agent to treat SCD and modulating PGC-1α activity or the signaling pathways that it regulates might benefit therapeutically patients with SCD.

The Association of SEPTIN6 Mutations with Pediatric Myelodysplasia

Septin family proteins belong to the RAS-like GTPase superclass and are considered as a structural element of the cytoskeleton. They are evolutionarily conserved and are important in cytokinesis, polarity, cell cycle, vesicle trafficking, and exocytosis. They are implicated in T and B cell lymphomas; and acute myeloid leukemias, where they frequently act as fusion partners with the Mixed-Lineage Leukemia (MLL) gene. Previously, we described two non-syndromic patients with severe neutropenia which progressed to myelodysplastic syndrome (MDS) (Renella et al. AJH, 2022, Mohamad et al. ASH, 2022). Next-generation sequencing revealed both patients (P1 and P2) to have novel X-linked germline mutations in codon 428 of the SEPTIN6 gene (P1: SEPTIN6 c.1282T>C; P2: SEPTIN6 c.1282T>A). One of these patients (P1) had an additional somatic stop-gain mutation (P1: SEPTIN6 c.43C>T) of unclear function. Both patient germline mutations were predicted to add 9 amino acids to the C-terminal end creating a novel mutated SEPTIN6 protein. Since SEPTIN6 is located on the X-chromosome and expression of these mutated proteins occurred in males, no wild-type protein was present in the patient cells. To further explore the biological relevance of these mutations, single base-pair gene editing was used to introduce the (P1: T>C) mutation into normal male donor CD34+ cells. Morphological studies demonstrated that SEPTIN6 edited cells had enlarged, multi-nucleated and dysplastic nuclei; and reduced clonogenic activity. To further assess the effect of the T>C mutation on hematopoietic stem cell (HSC) function, the SEPTIN6 edited cells were transplanted into immunocompromised NSGW mice. Analysis of peripheral blood demonstrated reduced chimerism in edited recipients (4.27% +/-3.4%) compared to controls (20.56% +/-6.2%, mean+/- SD, p= 0.0008) indicating a functional HSC defect upon mutation acquisition. Bulk mRNA sequencing demonstrated changes in ribosome, DNA replication, homologous recombination and mismatch repair pathways which imply DNA damage and cell cycle defects (Figure 1). To examine the potential dominant negative vs haploinsufficiency role of these mutations, we introduced the mutated SEPTIN6 (P1: SEPTIN6 c.1282T>C) cDNA into wild type human male CD34+ cells. Morphological studies demonstrated enlarged cells with multi-nucleated and dysplastic nuclei. Moreover, clonogenic assays demonstrated that cells containing both the mutated and wild type protein had reduced number of colonies (17.7+/- 5.5) compared to controls (127+/- 13.7, mean+/- SD, p <0.0001). This indicates that the mutated SEPTIN6 protein has a dominant negative effect over the wild type protein. We hypothesized that the somatic stop-gain mutation in the patient (P1) genome may mitigate the deleterious effects of the primary germline SEPTIN6 c.1282T>C mutation. To test this hypothesis, and determine the effect of each individual mutation, we created lentivirus vectors which contained a shmiR against the native SEPTIN6 mRNA and also contained (a) the germline P1: SEPTIN6 c.1282T>C mutation (M2 mutation) alone, or (b) the germline M2 mutation and the somatic stop-gain mutation (P1: M1+M2 mutation) in tandem, or (c) germline P2: SEPTIN6 c.1282T>A (M3 mutation). The wild type SEPTIN6 gene was reintroduced as a control. Clonogenic assays demonstrated that SEPTIN6 knockdown in human CD34+ cells caused no significant change in progenitor activity (Table 1). Furthermore, introduction of the M2 or the M3 germline mutants in SEPTIN6 knocked down cells caused a reduction in the total number of colonies, especially the number of GM colonies with no change in the number of BFU-E colonies (Table 1). This data validates our previous experiments performed with single base pair gene editing in human CD34+ cells. Interestingly, introduction of the stop-gain M1 mutation along with the M2 mutation (M1+M2) reversed the reduced clonogenic activity seen with expression of the M2 mutation (Table 1). This indicates that the M1 mutation by introducing an early stop codon creates a SEPTIN6 knockdown effect and rescues hematopoietic function from the deleterious M2 mutation. Overall, our data demonstrates the acquisition of de novo gain of function mutations in the SEPTIN6 gene in myelopoiesis and suggests deleterious effects on engrafting hematopoietic stem cells. These mutations are associated with the development of MDS in two pediatric patients.

Sgr-2921, a Potent CDC7 Inhibitor, Demonstrates Significant Anti-Leukemic Responses in Patient-Derived AML Models Representing Difficult-to-Treat Disease

Introduction: Elevated replication stress (RS) is a common feature of many highly proliferative cancers including AML. CDC7 has been shown to activate key components of the RS response, including ATR, and the BRCA1 and cohesin complexes, thereby mitigating accumulation of DNA damage and genomic instability. Inhibition of CDC7 in cancer cells results in an impaired response to RS, leading to accumulation of DNA damage and cell death. SGR-2921 is a potent inhibitor of CDC7 and a clinical trial to evaluate the safety, tolerability and preliminary antitumor activity of SGR-2921 in patients with AML and MDS is expected to begin in 2023 (NCT 05961839). We have previously shown that SGR-2921 has potent anti-proliferative activity in 12 out of 16 patient-derived AML samples ex vivo. Importantly, 6 out of 6 AML patient samples with mutations in TP53 responded to SGR-2921. Here we show that SGR-2921 results in potent, dose-dependent, anti-leukemic activity in multiple disseminated in vivo AML patient-derived xenograft (PDX) models representing difficult-to-treat disease. Methods: NOG-EXL mice were inoculated with primary human AML blast cells after sublethal irradiation. Engraftment of human AML blast cells were measured in surrogate mice by flow cytometry using human CD45 vs. mouse CD45 markers. When human CD45 engraftment reached >20% of bone marrow cells in surrogate mice, the study mice were randomized based on weight and dosed with vehicle, SGR-2921 at 3 dose levels or a standard of care agent. SGR-2921 was administered via oral gavage (PO) twice daily in cycles of 5 days of dosing followed by 9 days of no compound dosing over 2 cycles. In a separate study, phosphorylated MCM2 (a direct substrate of CDC7) was measured in the same cohort of mice engrafted with human AML cells following 3 doses of SGR-2921 to determine target engagement. Results: In vivo, SGR-2921 showed strong anti-leukemic activity in the AML PDX models at tolerated doses. Specifically, engrafted AML cells derived from a patient with TP53 mutated AML, a relapsed and refractory patient and a patient with KTM2a rearrangements showed significant and dose-dependent reduction in human CD45 positive AML blast cells. The reduction of blast cells was observed in both peripheral blood and bone marrow. The reduction in human CD45 positive blast cells were associated with an increase in mouse CD45 positive cells. In addition, SGR-2921 demonstrated dose-dependent inhibition of phosphorylated MCM2 in the spinal cord and spleen of the AML PDX models as surrogates of the bone marrow and extravascular compartments, respectively. Conclusions: Our data show remarkable dose-dependent in vivo activity of SGR-2921 in AML PDX models, including in those representing difficult-to-treat disease. Direct inhibition of CDC7 by SGR-2921 in AML blasts was demonstrated by a dose-dependent reduction of phosphorylated MCM2. Together, these data demonstrate that SGR-2921-mediated CDC7 inhibition is an attractive novel treatment opportunity in AML, with potential utility in patients with high risk mutations and relapsed and refractory AML.

Prime Editing Efficiently and Precisely Corrects Causative Mutation in Chronic Granulomatous Disease, Restoring Myeloid Function: Toward Development of a Prime Edited Autologous Hematopoietic Stem Cell Therapy

Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency characterized by a lack of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in phagocytic myeloid cells such as neutrophils. NADPH oxidase produces reactive oxygen species (ROS) that directly kill bacteria and fungi to control infection. CGD patients lack this ability to control infection, which results in chronic and severe infections, inflammation, autoimmunity, poor quality of life, and reduced lifespan. Current standard care for CGD is antimicrobial prophylaxis and aggressive management of infection. For patients with a suitable human leukocyte antigen (HLA) matched donor, an allogeneic hematopoietic stem cell (HSC) transplant (allo-HSCT) offers a curative approach. However, many patients do not have access to HLA matched donors and allo-HSCT also carries a risk of GvHD and graft rejection. P47phox-deficient CGD, the most common autosomal recessive CGD, is caused by mutations in the NCF1 gene, which encodes the p47phox protein, a subunit of the NADPH oxidase complex. Approximately 80% of p47phox-deficient CGD patients carry the same homozygous mutation, a 2 nucleotide GT deletion (delGT) in exon 2 of NCF1 which is correctable using Prime Editing. Transplantation of Prime Edited patient autologous CD34 + cells, in which the delGT mutation has been corrected with Prime Editing, may provide a curative treatment for p47phox-deficient CGD patients. To evaluate whether correction of the delGT NCF1 mutation in CGD patient CD34 + cells restores p47phox protein expression and NADPH oxidase activity in myeloid progeny, Prime Editors were designed to correct this mutation precisely and efficiently. The Prime Editors were delivered to CD34 + cells from two p47phox-deficient CGD patients homozygous for delGT NCF1. In both studies, ≧75% of Prime Edited patient CD34 + cells carried at least 1 corrected allele as determined by ddPCR and DNA sequencing analyses. Myeloid progeny differentiated from Prime Edited patient CD34 + cells had restored p47phox protein expression and NADPH oxidase activity to ~80% of normal healthy donors as determined by multiple ROS detection assays. To evaluate reproducibility, Prime Editors were delivered to CD34 + cells from 6 different healthy donors. On average, greater than 90% of CD34 + cells were Prime Edited across 6 donors in independent experiments. To confirm that Prime Edited CD34 + cells retain stem cell multipotency, engraftment, and hematopoietic reconstitution properties, Prime Edited and mock treated CD34 + cells from three different donors were transplanted into immunodeficient mice. Sixteen weeks after CD34 + cell transplantation, >87% of long-term hematopoietic stem cells (LT-HSC) that repopulated the bone marrow had at least 1 allele corrected. There were no significant differences between ‘electroporation only’ mock treated and Prime Editor electroporated human CD34 + cells with respect to long-term CD34 + cell engraftment, human CD45 + blood cell chimerism, hematopoietic multilineage blood cell reconstitution and biodistribution, or LT-HSC cell potency. Across these studies, >95% human CD45 + cell chimerism was achieved with Prime Edited CD34 + cells. Despite high on-target correction of the delGT mutation in NCF1, off-target editing, unintended editing, and chromosomal rearrangements (larger deletions or translocation) events were not detected using a robust suite of genome-wide assays. In summary, Prime Editing corrects a mutation that causes the most common autosomal recessive from of CGD; correction of this delGT mutation at NCF1 restores NADPH oxidase activity and function in myeloid progeny of Prime Edited CGD CD34 + cells; and Prime Edited long-term HSCs retain multipotency and functionality in vivo. These results support the development of a Prime Edited CGD patient autologous CD34 + cell therapy as a potential curative approach for p47phox-deficient CGD patients.