A Novel Aggressive NK Cell Leukemia Cell Line and Mouse Model for Evaluation of Therapeutic Candidates

Introduction: Aggressive nature killer-cell leukemia (ANKL) is a rare, highly lethal and disseminated malignancy derived from NK cells. Patients are most commonly young to middle aged adults who present with constitutional symptoms and often have organomegaly, hemophagocytosis, and multiorgan failure. Most cases are refractory to therapy and the median survival is less than 2 months despite multiagent chemotherapy. Development model systems and novel targeted therapeutic agents remains an urgent issue. In this study we established a cell line and a NOD-scid IL2Rgammanull(NSG) mouse model of ANKL, which represented the primary malignant cells. We characterized the molecular features of this model and explored new targeted agents against it. Methods: Primary tumor cells were cultured ex vivo in the presence of human IL-2 and then injected into a NSG mouse. Flow cytometry, immunohistochemistry (IHC), CISH-EBER were used to confirm the engraftment and to compare the pathologic features of engrafted tumor cells with the primary sample. Karyotyping and oligonucleotide/single nucleotide polymorphism microarray analyses were performed to compare the established cell line CCANKL with primary tumor cells. Selective inhibitors targeting anti-apoptotic BCL-2 family members and a CD6-targeted antibody drug conjugate (ADC) were tested for their treatment efficacies in this model. Results: Leukemic peripheral blood from a 31-year old woman with relapsed ANKL demonstrated a hyperleukocytosis in which NK cells accounted for 88% of cells. Immunophenotyping showed the malignant cells expressed CD2, 7, 38, 45 and 56, and lacked CD3, 4, 5, 8, 10, 11b, 13, 14, 16, 19, 20, 22, 33, 34, 57, 64, 65, 117, HLADR and Kappa or Lambda immunoglobulin light chain. IHC of an involved lymph node showed the presence of EBV (EBER in situ hybridization) and a proliferative index of 70%. Isolated PBMC were cultured ex vivo and serially passaged as a cell line (CCANKL) in the presence of human IL2. CCANKL cells successfully engrafted in NSG mice and displayed splenomegaly. IHC and Immunophenotyping data confirmed both CCANKL cells and engrafted cells in NSG mice represented primary tumor cells. Both the primary PBMC and CCANKL cells had similar, abnormal karyotypes of 46,XX,dup(1)(q21q42), del(6)(q15q25)[1]/46,idem,der(20)t(7;20)(q11.2;q13.1)[9] and 46,XX,del(6)(q15q25),der(20)t(7;20)(q11.2;q13.1),add(22)(p11.1)[10], respectively. Microarray analysis of both primary tumor cells and CCANKL confirmed some of the karyotypic findings, but also revealed additional abnormalities, in particular for the CCANKL cell line. There were also some differences for the copy number changes and regions of loss of heterozygosity observed between the primary tumor cells and CCANKL. Abnormalities involving 1q, 6q and 7p have been reported in association with aggressive NK cell leukemia. Both immunoblotting and IHC revealed expression of anti-apoptotic proteins of BCL-xL, MCL-1 and pro-apoptotic BIM in CCANKL cells and NSG-engrafted tumors, while BCL-2 was positive in cultured cells by Western blotting but not detectable in engrafted tissue via IHC. Expression level of BCL-xl was stable but there were enhanced expression of MCL-1 and BIM during 72-hour starvation of IL2 in cultured CCANKL cells. Single agent of venetoclax, BCL-xl inhibitor (A-1331852), and CDK9 inhibitor (A-1592668), at range of 1 - 1000 nM, had no detectable (BCL-2), mild (BCL-xL), and strong (CDK9) killing activities against CCANKL cells. We further confirmed expression of CD6, a surface glycoprotein expressed on most T-cells and a subset of NK cells, by IHC and flow cytometry in CCANKL and also tested a novel CD6-targeted ADC. Compared to naked anti-CD6 IgG and isotype control, CD6-ADC was capable of inducing apoptosis in CCANKL cells. Administration of either anti-CD6 IgG or CD6-ADC (4 mg/kg) on the same day as inoculation of CCANKL cells in NSG mice blocked engraftment of these malignant cells in NSG mice. The 2 nd ANKL case tested by IHC showed positive of CD6 in malignant cells. Conclusion: A novel aggressive NK cell leukemia cell line and a NSG mouse model were established. The in vitro data support further investigation of the expression features of BCL-2 family proteins in ANKL cases and in vivo evaluation for the efficiency of anti-apoptotic protein inhibitors in this aggressive disease. Further studies targeting CD6 in ANKL is warranted. Disclosures Lin: Takeda Pharma: Consultancy, Research Funding. Phillips: AbbVie Inc.: Current Employment, Current equity holder in publicly-traded company. Hsi: AbbVie Inc, Eli Lilly: Research Funding.

mSOD1-NSG mice: a new in vivo model to test human T cells in ALS

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease with unclear etiology and few treatment options. Engineering of human cells for therapy has great potential to provide a means to create long-lived therapeutics that can respond to local signals within tissues. One challenge for development of human cell therapeutics is to have disease models that do not reject transplanted human cells. G93A mutant superoxide dismutase-1 (mSOD1) transgenic mouse model is a robust disease model for ALS, with development of local inflammation, activation of microglia and astrocytes, motor neuron death, and development of paralysis. To create a mouse model for ALS that permits the transplantation of human T cells without immune-mediate rejection, we bred the G93A transgene onto the NOD-SCID-IL-2Rγ-deficient (NSG) mouse model to create mSOD1-NSG mice. We report that mSOD1-NSG mice develop a progressive ALS-like disease with microgliosis, astrogliosis, and paralysis development with an average onset at 11 weeks and end-stage at 14 weeks. Transplanted human T regulatory cells survive in these mice for at least 60 days. This mSOD1-NSG mouse model for ALS can be used to test human T cell-based therapeutics, and it may be helpful to test any human cell-based therapy for ALS.

An Improved Animal Model of Multiple Myeloma Bone Disease

Multiple myeloma (MM) is a plasma cell malignancy that causes an accumulation of terminally differentiated monoclonal plasma cells in the bone marrow, accompanied by multiple myeloma bone disease (MMBD). MM animal models have been developed and enable to interrogate the mechanism of MM tumorigenesis. However, these models demonstrate little or no evidence of MMBD. We try to establish the MMBD model with severe bone lesions and easily accessible MM progression. 1 × 106 luciferase-expressing 5TGM1 cells were injected into 8–12 week-old NOD SCID gamma mouse (NSG) and C57BL/KaLwRij mouse via the tail vein. Myeloma progression was assessed weekly via in vivo bioluminescence (BL) imaging using IVIS-200. The spine and femur/tibia were extracted and scanned by the micro-computer tomography for bone histo-morphometric analyses at the postmortem. The median survivals were 56 days in NSG while 44.5 days in C57BL/KaLwRij agreed with the BL imaging results. Histomorphic and DEXA analyses demonstrated that NSG mice have severe bone resorption that occurred at the lumbar spine but no significance at the femur compared to C57BL/KaLwRij mice. Based on these, we conclude that the systemic 5TGM1 injected NSG mouse slowly progresses myeloma and develops more severe MMBD than the C57BL/KaLwRij model.

The antimalarial MMV688533 provides potential for single-dose cures with a high barrier to Plasmodium falciparum parasite resistance

The emergence and spread of Plasmodium falciparum resistance to first-line antimalarials creates an imperative to identify and develop potent preclinical candidates with distinct modes of action. Here, we report the identification of MMV688533, an acylguanidine that was developed following a whole-cell screen with compounds known to hit high-value targets in human cells. MMV688533 displays fast parasite clearance in vitro and is not cross-resistant with known antimalarials. In a P. falciparum NSG mouse model, MMV688533 displays a long-lasting pharmacokinetic profile and excellent safety. Selection studies reveal a low propensity for resistance, with modest loss of potency mediated by point mutations in PfACG1 and PfEHD. These proteins are implicated in intracellular trafficking, lipid utilization, and endocytosis, suggesting interference with these pathways as a potential mode of action. This preclinical candidate may offer the potential for a single low-dose cure for malaria.

Thymectomy procedure to remove native thymus of NSG mice v1

This protocol details our approach to removing the native thymus from an NSG mouse. The NSG mouse thymus is atrophied, but through experiments in our lab, we have found that these thymi are still functional and capable of producing CD3+ cells. Removal of these native thymi prevents the production of CD3+ cells in NSG mice if a replacement thymus is not implanted. Due to their atrophic state, the native thymi are difficult to visualize during surgery and much practice is required to reduce mortality during the operation.

A General Guide To Generate Different Humanized Mouse Models v1

This protocol details how to create humanized mouse models from NSG mice. Four different variants of humanized mice can be generated based on whether or not the native thymus is retained or if a human thymus piece is transplanted. Which type of humanized mouse is desired depends on the goals for the experiment. See our protocols on NSG mouse thymectomy, human CD34+ cell isolation, and human fetal thymus preparation for more details on some of the steps in this protocol (“Thymectomy procedure to remove native thymus of NSG mice”, “Human CD34+ cell isolation from fetal liver, and fetal thymus preparation”, “CD34+ isolation from human bone marrow”).

An animal model of MGUS/SMM to investigate the role of cellular senescence in progression to MM.

8050 Background: Multiple Myeloma (MM) is an incurable cancer of plasma cells that arises from precursor stages, monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering MM (SMM). Mechanisms for the transformation of MGUS/SMM to MM are not fully known. There is no approved therapy for MGUS/SMM and research is limited by a lack of animal models. Here, we describe the use of a novel animal model of disease progression derived from patients with MGUS/SMM to study the impact of cellular senescence (CS) on the development of MM. Methods: CD 138+ plasma cells and CD138- stromal cells were isolated via magnetic beads from bone marrow aspirates of patients with MGUS/SMM and co-cultured CD138+/CD138- with different ratios. For the animal model CD138+/CD138- cells from MGUS/SMM patients were injected into the tibias of NSG mice (1:10). After 4 weeks, CD138+ cells were harvested and again co-transplanted into the tibia of a 2nd NSG mouse with the CD138- cells (1:10). Mice underwent serial imaging and tibias were histologically examined after 4 weeks in the 2nd mouse. CS gene expression was compared by RNAseq between patients with SMM and healthy older adults. The primary CD138- cells were stained for SA-β-gal. Human mesenchymal stem cells (hMSCs) induced by H2O2 for CS vs control were co-cultured with CD138+ cells. Patient derived CD138- cells were treated with anti-senolytic drugs, dasatinib (D) and quercetin (Q) and cell growth in co-culture with CD138+ plasma cells was measured. D+Q treated patient-derived CD138+/CD138- cells were co-transplanted into our NSG mouse model and followed with serial imaging. Results: CD138- stromal cells from patients with MGUS/SMM support the growth of CD138+ plasma cells (10:1). CD138- cells were found to gain CD138+ expression, suggesting another source for the plasma cell growth. Imaging of our mouse model showed the development of lytic lesions in the tibias of 5/5 mice versus no lytic lesions in mice transplanted with CD138+ cells alone. Staining of the lytic lesions revealed CD138+ plasma cells. Our RNA seq showed significantly increased expression of CS genes, CDKN2A, p16 and p19 (3 fold) in CD138- cells of SMM patients and confirmed by qPCR (8 fold) compared to healthy older adults. The primary stromal cell culture from MGUS/SMM patients showed the presence of SCs by SA-β-gal staining. H2O2 induced senescent hMSCs stimulate cell survival and growth of CD138+ cells. CD138- cells cultured with conditioned media from H2O2 induced hMSCs demonstrated more CD138+ cells than vehicle treated CD138- cells. Conversely, D + Q pre-treated CD138- cells lost the ability to promote CD138+ cells in co-culture. Our animal model treated with D+Q showed less bone erosion in tibias compared to untreated mice. Conclusions: Our work demonstrates a novel animal model for studying disease progression in patients with MGUS or SMM and demonstrates a role for CS in MM disease progression.

Extramedullary multiple myeloma patient derived orthotopic xenograft with high disturbed genome: combined exhaustive molecular and therapeutic studies

Extramedullary multiple myeloma (EMM) has an overall survival of 6 months and occurs in 20% of multiple myeloma (MM) patients. Genetic and epigenetic mechanisms involved in EMM and the therapeutic role of new agents for MM are not well established. Besides, well characterized preclinical models for EMM are not available. Herein, a patient derived orthotopic xenograft (PDOX) was generated from a patient with an aggressive EMM to study in-depth genetic and epigenetic events and drug responses related to extramedullary disease. A fresh punch of an extramedullary cutaneous lesion was orthotopically implanted in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ(NSG) mouse. The PDOX mimicked histologic and phenotypic features of patient's tumor. Cytogenetic studies revealed a hyperploid genome with multiple genetic poor-prognosis alterations. Copy number alterations were detected in all chromosomes. The IGH translocation t(14;16)(q32;q23)IGH/MAF were already observed at medullary stage and a new one, the t(10;14)(p?11-12;q32), were observed only at extramedullary disease and could be eventually related to EMM progression in this case. Exome sequencing showed 24 high impact SNV and 180 indels. From the genes involved, only TP53 was previously described as a driver in MM. A rather balanced proportion of hyper/hypomethylated sites different to previously reported widespread hypomethylation in MM was also observed. Treatment with lenalidomide, dexamethasone and carfilzomib showed a tumor weight reduction of 90% vs. non-treated tumors while the anti-CD38 antibody Daratumumab showed a reduction of 46%. The generation of PDOX from small EMM biopsy allowed to go in-deep to the molecular events associated to extramedullary disease in combination with drug testing.

Development of a Novel Human CD147 Transgenic NSG Mouse Model to test SARS-CoV-2 Infection and Immune Responses

An animal model that can mimic the SARS-CoV-2 infection in humans is critical to understanding the newly emerged, rapidly spreading SARS-CoV-2 and development of therapeutic strategies. Studies show that the spike (S) proteins of SARS-CoV (SARS-CoV-S-1-S) and SARS-CoV-2 (SARS-CoV-2-S) bind to human angiotensin-converting enzyme 2 (hACE2, a well-recognized, functional receptor for SARS-CoV and SARS-CoV-2) to mediate viral entry. Several hACE2 transgenic (hACE2Tg) mouse models are being widely used, which is clearly invaluable. However, the hACE2Tg mouse model cannot fully explain: 1) low expression of ACE2 observed in human lung and heart, but lung or heart failure occurs frequently in severe COVID-19 patients); 2) low expression of ACE2 on immune cells, but lymphocytopenia occurs frequently in COVID-19 patients; and 3) hACE2Tg mice do not develop strong clinical disease following SARS-CoV-2 infection in contrast to SARS-CoV-1. Moreover, one of most outstanding features of coronaviruses is the diversity of receptor usage, which includes the newly proposed human CD147 (hCD147) as a receptor for SARS-CoV-2-S. It is still debatable whether CD147 can serve as a functional receptor for SARS-CoV-2 infection or entry. Here we successfully generated a hCD147Tg mouse model in the NOD-scid IL2Rgammanull (NSG) background. In this hCD147Tg-NSG mouse model, the hCD147 genetic sequence was placed following the endogenous mouse promoter for mouse CD147 (mCD147), which creates an in vivo model that may better recapitulate physiological expression of CD147 proteins at the molecular level compared to the existing and well-studied K18-hACE2-B6 model. In addition, the hCD147Tg-NSG mouse model allows further study of SARS-CoV-2 in the immunodeficiency condition which may assist our understanding of this virus in the context of high-risk populations with immunosuppressed conditions. The hCD147Tg-NSG mouse mode can serve as an additional animal model for interrogate whether CD147 serve as an independent functional receptor or accessory receptor for SARS-CoV-2 entry and immune responses.