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Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional ...

Medical University of Graz

The chimeric antigen receptor T cell (CAR-T cell) immunotherapy currently represents a hot research trend and it is expected to revolutionize the field of cancer therapy. Promising outcomes have been achieved using CAR-T cell therapy for haematological malignancies. Despite encouraging results, several challenges still pose eminent hurdles before being fully recognized. Directing CAR-T cells to target a single tumour associated antigen (TAA) as the case in haematological malignancies might be much simpler than targeting the extensive inhibitory microenvironments associated with solid tumours. This review focuses on the basic principles involved in development of CAR-T cells, emphasizing the differences between humoral IgG, T-cell receptors, CAR and Fcγ-CR constructs. It also highlights the complex inhibitory network that is usually associated with solid tumours, and tackles recent advances in the clinical studies that have provided great hope for the future use of CAR-T cell immunotherapy. While current Fcγ-CR T cell immunotherapy is in pre-clinical stage, is expected to provide a sound therapeutic approach to add to existing classical chemo- and radio-therapeutic modalities.

Recent perspective on CAR and Fcγ-CR T cell immunotherapy for cancers: Preclinical evidence versus clinical outcomes.

KRAS mutations hinder therapeutic efficacy of epidermal growth factor receptor (EGFR)-specific monoclonal antibodies cetuximab and panitumumab-based immunotherapy of EGFR+ cancers. Although cetuximab inhibits KRAS-mutated cancer cell growth in vitro by natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), KRAS-mutated colorectal carcinoma (CRC) cells escape NK cell immunosurveillance in vivo. To overcome this limitation, we used cetuximab and panitumumab to redirect Fcγ chimeric receptor (CR) T cells against KRAS-mutated HCT116 colorectal cancer (CRC) cells. We compared four polymorphic Fcγ-CR constructs including CD16 -CR, CD16 -CR, CD32 -CR, and CD32 -CR transduced into T cells by retroviral vectors. Percentages of transduced T cells expressing CD32 -CR (83.5 ± 9.5) and CD32 -CR (77.7 ± 13.2) were significantly higher than those expressing with CD16 -CR (30.3 ± 10.2) and CD16 -CR (51.7 ± 13.7) (p < 0.003). CD32 -CR T cells specifically bound soluble cetuximab and panitumumab. However, only CD16 -CR T cells released high levels of interferon gamma (IFNγ = 1,145.5 pg/ml ±16.5 pg/ml, p < 0.001) and tumor necrosis factor alpha (TNFα = 614 pg/ml ± 21 pg/ml, p < 0.001) upon incubation with cetuximab-opsonized HCT116 cells. Moreover, only CD16 -CR T cells combined with cetuximab killed HCT116 cells and A549 KRAS-mutated cells in vitro. CD16 -CR T cells also effectively controlled subcutaneous growth of HCT116 cells in CB17-SCID mice in vivo. Thus, CD16 -CR T cells combined with cetuximab represent useful reagents to develop innovative EGFR+KRAS-mutated CRC immunotherapies.

CD16-158-valine chimeric receptor T cells overcome the resistance of KRAS-mutated colorectal carcinoma cells to cetuximab.

Cetuximab and panitumumab bind the human epidermal growth factor receptor (EGFR). Although the chimeric cetuximab (IgG1) triggers antibody-dependent-cellular-cytotoxicity (ADCC) of EGFR positive target cells, panitumumab (a human IgG2) does not. The inability of panitumumab to trigger ADCC reflects the poor binding affinity of human IgG2 Fc for the FcγRIII (CD16) on natural killer (NK) cells. However, both human IgG1 and IgG2 bind the FcγRII (CD32A) to a similar extent. Our study compares the ability of T cells, engineered with a novel low-affinity CD32A -chimeric receptor (CR), and those engineered with the low-affinity CD16 -CR T cells, in eliminating EGFR positive epithelial cancer cells (ECCs) in combination with cetuximab or panitumumab. After T-cell transduction, the percentage of CD32A -CR T cells was 74 ± 10%, whereas the percentage of CD16 -CR T cells was 46 ± 15%. Only CD32A -CR T cells bound panitumumab. CD32A -CR T cells combined with the mAb 8.26 (anti-CD32) and CD16 -CR T cells combined with the mAb 3g8 (anti-CD16) eliminated colorectal carcinoma (CRC), HCT116 cells, in a reverse ADCC assay in vitro. Crosslinking of CD32A -CR on T cells by cetuximab or panitumumab and CD16 -CR T cells by cetuximab induced elimination of triple negative breast cancer (TNBC) MDA-MB-468 cells, and the secretion of interferon gamma and tumor necrosis factor alpha. Neither cetuximab nor panitumumab induced Fcγ-CR T antitumor activity against Kirsten rat sarcoma (KRAS)-mutated HCT116, nonsmall-cell-lung-cancer, A549 and TNBC, MDA-MB-231 cells. The ADCC of Fcγ-CR T cells was associated with the overexpression of EGFR on ECCs. In conclusion, CD32A -CR T cells are efficiently redirected by cetuximab or panitumumab against breast cancer cells overexpressing EGFR.

In vitro elimination of epidermal growth factor receptor-overexpressing cancer cells by CD32A-chimeric receptor T cells in combination with cetuximab or panitumumab.

Epidermal hyperplasia represents a morphologic hallmark of psoriatic skin lesions. Langerhans cells (LCs) in the psoriatic epidermis engage with keratinocytes (KCs) in tight physical interactions; moreover, they induce T-cell-mediated immune responses critical to psoriasis.

BMP7 aberrantly induced in the psoriatic epidermis instructs inflammation-associated Langerhans cells.

Bone morphogenetic proteins (BMPs) are members of the TGF-β family that signal via the BMP receptor (BMPR) signaling cascade, distinct from canonical TGF-β signaling. BMP downstream signaling is strongly induced within epidermal keratinocytes in cutaneous psoriatic lesions, and BMP7 instructs monocytic cells to acquire characteristics of psoriasis-associated Langerhans dendritic cells (DCs). Regulatory T (Treg)-cell numbers strongly increase during psoriatic skin inflammation and were recently shown to limit psoriatic skin inflammation. However, the factors mediating Treg-cell accumulation in psoriatic skin currently remain unknown.

Bone morphogenetic protein signaling regulates skin inflammation via modulating dendritic cell function.

Gene profiling revealed that the S1P signaling pathway is induced by TGF-β1 during LC commitment of monocytopoietic cells. Constitutive-active TGF-β1-S1P signaling seems to elevate the activation threshold of LCs and thereby prevent inappropriate and overshooting immune responses to microbial and physicochemical environmental signals. In turn, signals that lead to LC migration may disrupt this pathway via inhibiting S1P bioavailability.

Induction of the sphingosine-1-phosphate signaling pathway by TGF-β1 during Langerhans-type dendritic cell differentiation.

Transforming growth factor β (TGF-β) family ligands are key regulators of dendritic cell (DC) differentiation and activation. Epidermal Langerhans cells (LCs) require TGF-β family signaling for their differentiation, and canonical TGF-β1 signaling secures a non-activated LC state. LCs reportedly control skin inflammation and are replenished from peripheral blood monocytes, which also give rise to pro-inflammatory monocyte-derived DCs (moDCs). By studying mechanisms in inflammation, we previously screened LCs versus moDCs for differentially expressed microRNAs (miRNAs). This revealed that miR-424/503 is the most strongly inversely regulated (moDCs > LCs). We here demonstrate that miR-424/503 is induced during moDC differentiation and promotes moDC differentiation in human and mouse. Inversely, forced repression of miR-424 during moDC differentiation facilitates TGF-β1-dependent LC differentiation. Mechanistically, miR-424/503 deficiency in monocyte/DC precursors leads to the induction of TGF-β1 response genes critical for LC differentiation. Therefore, the miR-424/503 gene cluster plays a decisive role in anti-inflammatory LC versus pro-inflammatory moDC differentiation from monocytes.

The miR-424(322)/503 gene cluster regulates pro- versus anti-inflammatory skin DC subset differentiation by modulating TGF-β signaling.

Bone morphogenetic proteins (BMPs) are potent signaling molecules initially described as osteopromoting proteins. BMPs represent one of the members of the larger TGFβ family and today are recognized for their important role in numerous processes. Among the wide array of functions recently attributed to them, BMPs were also described to be involved in the regulation of components of the innate and adaptive immune response. This review focuses on the signaling pathway of BMPs and highlights the effects of BMP signaling on the differentiation, activation, and function of the main cell types of the immune system.

Regulation of the Immune System in Health and Disease by Members of the Bone Morphogenetic Protein Family.

Acute myeloid leukemia (AML) is a malignant disease of hematopoietic precursors at the earliest stage of maturation, resulting in a clonalproliferation of myoblasts replacing normal hematopoiesis. AML represents one of the most common types of leukemia, mostly affecting elderly patients. To date, standard chemotherapy protocols are only effective in patients at low risk of relapse and therapy-related mortality. The average 5-year overall survival (OS) is approximately 28%. Allogeneic hematopoietic stem cell transplantation (HSCT) improves prognosis but is limited by donor availability, a relatively young age of patients, and absence of significant comorbidities. Moreover, it is associated with significant morbidity and mortality. However, increasing understanding of AML immunobiology is leading to the development of innovative therapeutic strategies. Immunotherapy is considered an attractive strategy for controlling and eliminating the disease. It can be a real breakthrough in the treatment of leukemia, especially in patients who are not eligible forintensive chemotherapy. In this review, we focused on the progress of immunotherapy in the field of AML by discussing monoclonal antibodies (mAbs), immune checkpoint inhibitors, chimeric antigen receptor T cells (CAR-T cells), and vaccine therapeutic choices.

Immunotherapy as a Turning Point in the Treatment of Acute Myeloid Leukemia.

Dietary polyphenols, which are present in  have been associated with various beneficial effects on human health including antioxidant, antiviral, and anti-inflammatory activities. We aimed to investigate the immunomodulatory effects of aronia juice polyphenols in a randomized placebo-controlled human intervention study and cell culture experiments. A total of 40 females were asked to consume either 200 mL of aronia juice or a placebo drink for six weeks and were investigated again after a washout period of another six weeks. We observed that only half of the participants tolerated the aronia juice well (Vt) and the other half reported complaints (Vc). The placebo (P) was generally tolerated with one exception ( = 0.003). Plasma polyphenol levels increased significantly in Vt after the intervention ( = 0.024) but did neither in P nor in Vc. Regulatory T cell (Treg) frequencies remained constant in Vt and P during the intervention, whereas Tregs decreased in Vc ( = 0.018). In cell culture, inhibiting effects of ferulic acid ( = 0.0005) and catechin ( = 0.0393) on the differentiation of Tregs were observed as well as reduced activation of CD4-T cells in ferulic acid ( = 0.0072) and aronia juice ( = 0.0163) treated cells. Interestingly, a CD4CD25FoxP3 cell population emerged in vitro in response to aronia juice, but not when testing individual polyphenols. In conclusion, our data strengthen possible individual hormetic effects, the importance of the food matrix for bioactivity, and the need for further investigations on possible impacts of specific physiological features such as the gut microbiota in the context of personalized nutrition.

Immunomodulatory Effects of Aronia Juice Polyphenols-Results of a Randomized Placebo-Controlled Human Intervention Study and Cell Culture Experiments.

The FcγRII (CD32) ligands are IgFc fragments and pentraxins. The existence of additional ligands is unknown. We engineered T cells with human chimeric receptors resulting from the fusion between CD32 extracellular portion and transmembrane CD8α linked to CD28/ζ chain intracellular moiety (CD32-CR). Transduced T cells recognized three breast cancer (BC) and one colon cancer cell line among 15 tested in the absence of targeting antibodies. Sensitive BC cell conjugation with CD32-CR T cells induced CD32 polarization and down-regulation, CD107a release, mutual elimination, and proinflammatory cytokine production unaffected by human IgGs but enhanced by cetuximab. CD32-CR T cells protected immunodeficient mice from subcutaneous growth of MDA-MB-468 BC cells. RNAseq analysis identified a 42 gene fingerprint predicting BC cell sensitivity and favorable outcomes in advanced BC. ICAM1 was a major regulator of CD32-CR T cell-mediated cytotoxicity. CD32-CR T cells may help identify cell surface CD32 ligand(s) and novel prognostically relevant transcriptomic signatures and develop innovative BC treatments.

Direct CD32 T-cell cytotoxicity: implications for breast cancer prognosis and treatment.

Calreticulin (CALR) mutations present the main oncogenic drivers in JAK2 wildtype (WT) myeloproliferative neoplasms (MPN), including essential thrombocythemia and myelofibrosis, where mutant (MUT) CALR is increasingly recognized as a suitable mutation-specific drug target. However, our current understanding of its mechanism-of-action is derived from mouse models or immortalized cell lines, where cross-species differences, ectopic over-expression and lack of disease penetrance are hampering translational research. Here, we describe the first human gene-engineered model of CALR MUT MPN using a CRISPR/Cas9 and adeno-associated viral vector-mediated knock-in strategy in primary human hematopoietic stem and progenitor cells (HSPCs) to establish a reproducible and trackable phenotype in vitro and in xenografted mice. Our humanized model recapitulates many disease hallmarks: thrombopoietin-independent megakaryopoiesis, myeloid-lineage skewing, splenomegaly, bone marrow fibrosis, and expansion of megakaryocyte-primed CD41 progenitors. Strikingly, introduction of CALR mutations enforced early reprogramming of human HSPCs and the induction of an endoplasmic reticulum stress response. The observed compensatory upregulation of chaperones revealed novel mutation-specific vulnerabilities with preferential sensitivity of CALR mutant cells to inhibition of the BiP chaperone and the proteasome. Overall, our humanized model improves purely murine models and provides a readily usable basis for testing of novel therapeutic strategies in a human setting.

Tommaso Sconocchia

Division of Hematology,
Department of Internal Medicine,
Division of Hematology, Department of Internal Medicine

Medical University of Graz

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Tommaso Sconocchia

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Medical University of Graz

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Division of Hematology,
Department of Internal Medicine,
Division of Hematology, Department of Internal Medicine