key: cord-0730184-bqb2y5bp
authors: Strich, Jeffrey R.; Kanthi, Yogendra
title: VITT(al) insights into vaccine-related clots
date: 2021-12-02
journal: Blood
DOI: 10.1182/blood.2021014195
sha: aebd73004c29dc1f4b753bedf903be06f092b0fd
doc_id: 730184
cord_uid: bqb2y5bp

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maturational processing, trafficking, membrane localization, signaling interactions, and stability. Several cancer-associated proteins are known to be palmitoylated, a classic example being the RAS family of small GTPases, where palmitoylation dictates trafficking, membrane localization, and signaling properties. 8, 9 However, the role of palmitoylation in regulating FLT3-ITD localization and signaling has not been previously shown. Lv et al elegantly demonstrate that S-palmitoylation mediated by ZDHHC6 plays a critical role in determining FLT3-ITD localization and activity (see figure) . They show that disruption of palmitoylation promotes trafficking of FLT3-ITD from the ER to the plasma membrane, and leads to activation of AKT and ERK while still maintaining activation of STAT5, and thereby increased FLT3-ITD-mediated leukemic progression. In contrast, palmitoylation did not play a significant role in trafficking of FLT3-WT and TKD mutant proteins to the plasma membrane or their signaling or cellular effects. They further confirmed that FLT3 proteins were palmitoylated, and that ZDHHC6-mediated palmitoylation regulated FLT3-ITD surface expression, signaling and growth in primary human FLT3-ITD 1 AML cells.

It is of note that FLT3-ITD phosphorylation did not affect palmitoylation, and that TKI treatment further increased the surface levels of a palmitoylation-deficient ITD mutant, suggesting that palmitoylation and phosphorylation are separate mechanisms regulating FLT3-ITD intracellular localization. The relationship of palmitoylation to receptor glycosylation and maturation was not evaluated, and requires further study. Palmitoylation-deficient FLT3-ITD mutants retained sensitivity to gilteritinib. Importantly, pharmacological inhibition of FLT3-ITD depalmitoylation using a pandepalmitoylase inhibitor significantly reduced FLT3-ITD surface expression, inhibited AKT and ERK signaling, and reduced cell growth. The depalmitoylase inhibitor synergized with Gilteritinib in inhibiting FLT3-ITD surface localization, AKT and ERK signaling, and abrogating growth of primary FLT3-ITD 1 AML cells. These observations provide new insights into the role of lipid modifications in compartmentalization of FLT3-ITD signaling in AML. Importantly, they indicate that targeting of depalmitoylation could be a potential therapeutic strategy for FLT3-ITD 1 leukemias and support further exploration and development of clinically applicable inhibitors of depalmitoylation. Because resistance to gilteritinib has been associated with reactivation of RAS/MAPK pathway, it will be of interest to determine whether depalmitoylation inhibitors provide additional benefit in FLT3-ITD 1 AML through inhibition of RAS/MAPK signaling. 3 The implications of these studies extend beyond FLT3-ITD AML because subcellular localization is a general mechanism that affects activation of RTKs and their downstream pathways. 7 Abnormal maturation and trafficking have also been observed for other oncogenic RTKs, which in addition to being aberrantly active in different cellular compartments, can also generate different signaling outputs depending on localization. The role of palmitoylation in localization, signaling, and transforming activity of other RTKs will doubtless be the subject of future studies. The authors also observed that serum from patients with VITT robustly initiated platelet aggregation when presented with PF4. Notably, this effect was fully abrogated by blockade of the FcgIIa receptor (FcgRIIA). Prothrombotic neutrophil extracellular traps (NETs), known to occur in acute COVID 5 and in autoimmune heparin-induced thrombocytopenia (HIT), also formed when neutrophils were stimulated with VITT serum or affinity purified anti-PF4 IgG in the presence of PF4 and platelets. Greinacher et al and another recent report 6 observed that NETs were more prevalent in CVST tissue from patients with VITT compared with VITT-unrelated CVST.

Taken together, these data support the hypothesis that VITT pathogenesis occurs in a 2-step process (see figure) . In the first step, shortly after vaccine inoculation, vaccine components and PF4 form neoantigens, promoting a proinflammatory vascular milieu that amplifies the adaptive immune response including production of anti-PF4 antibodies. In the second step, 1 to 3 weeks after inoculation, complexes of polyanion/PF4/anti-PF4 antibody activate neutrophils and platelets in an FcgRIIA-dependent manner, leading to thrombosis accretion in atypical vascular beds. Because the number of patients diagnosed with VITT is low, it remains unclear whether VITT has a predilection for thrombosis in unusual sites such as the cerebral venous sinus or whether thromboses in more typical sites such as the peripheral veins do not raise the clinical alarm to trigger diagnostic testing.

These findings provide detailed insight to VITT pathogenesis and the reasons that it may occur after vaccination with adenoviral vector-based vaccines but not mRNAbased vaccines. However, many questions remain. What is the protein(s) in the vaccine that binds to PF4? What is the precise neoantigen generated when PF4 and vaccine components interact? Do human proteins in the vaccine provoke an immune response? Is the prothrombotic antibody repertoire in VITT limited to PF4, the vaccine and its components, or is there overlap with autoantibodies found in acute COVID, autoimmune disease, and other critical illnesses? What is the half-life of immune complexes or the effect of multiple vaccine doses on the autoantibody response? Ultimately, answers to these and other questions will be needed to inform future development of vaccines and therapeutics that use adenovirusand other virus-based vectors.

Step 1 (days) Adenoviral vaccine EDTA Immune activation

Step As more is understood about the molecular disruptions that occur during VITT, the question remains; what is the best treatment for patients? Proposed therapeutics include IV immunoglobulin (IVIG) because of its success in treating autoimmune HIT, nonheparin anticoagulants, and plasmapheresis. A small body of evidence from nonrandomized trials and retrospective studies suggests that IVIG may be an effective treatment of VITT, although sometimes incomplete. 7 The results of this study by Greinacher et al bring to light another potential therapeutic, the spleen tyrosine kinase (SYK) inhibitor, fostamatinib that is currently used for the treatment of chronic immune thrombocytopenia. Fostamatinib inhibits activation of Fc receptor by antigen/antibody complexes, and reduced NETosis and platelet activation in ex vivo COVID studies. 8 In hospitalized patients with COVID, where circulating, prothrombotic antibodies that activate neutrophils, platelets, and endothelium have been identified, 9 orally administered fostamatinib reduced adverse events and showed a trend toward clinical benefit. 10 Although large, randomized trials are impractical in VITT given its low incidence, the FcgRIIAdependent signaling mechanism leading to platelet activation in VITT identified by Greinacher et al provides strong rationale to consider SYK inhibition in the limited therapeutic armamentarium of clinicians treating VITT and perhaps other forms of autoantibody-mediated thrombosis.

Conflict-of-interest disclosure: Y.K. serves as a board member for the Society for Vascular Medicine, participates in the National Heart, Lung and Blood Institute (NHLBI) CON-NECTS program and ACTIV-4 Host Tissue trial, and is an author on an unrelated patent application by the University of Michigan for the use of biogases in vascular disease. J.R.S. was the principal investigator of a clinical trial sponsored by the NHLBI to evaluate fostamatinib in acute COVID and participates in the ACTIV-4 Host Tissue trial. n R E F E R E N C E S

Insights in ChAdOx1 nCoV-19 vaccineinduced immune thrombotic thrombocytopenia

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK

Thrombotic thrombocytopenia after ChAdOx1 nCov-19 vaccination

Heparin-induced thrombocytopenia: a historical perspective

Neutrophil extracellular traps in COVID-19

Immune complexes, innate immunity, and NETosis in ChAdOx1 vaccine-induced thrombocytopenia

Cooling down VITT with IVIG

ROCKin' cGVHD treatment: has the time come?

In this issue of Blood, Cutler et al 1 report encouraging results from a randomized, multicenter, phase 2 trial (the ROCKstar Study) of treatment with the ROCK2 inhibitor belumosudil in patients with inadequately controlled chronic graft-versus-host disease (cGVHD) after 2 or more lines of prior therapy.When dire diseases are cured by allogeneic hematopoietic cell transplantation, patients still face many obstacles in their struggle to return to normal. Among them, cGVHD is a leading cause of nonrelapse mortality and morbidity. Between 35% and 70% of patients develop cGVHD with 30% to 50% of them having steroidrefractory or steroid-dependent cGVHD. After starting initial systemic therapy for National Institute of Health (NIH)-defined moderate or severe cGVHD, only 1 of 3 patients will be alive and off immunosuppression 5 years later. 2 This significant burden of GVHD in survivors has led to the introduction of a composite end point of cGVHD plus relapse-free survival. Manifestations of cGVHD are heterogenous and affect multiple organs. Among them, keratoconjunctivitis sicca, sclerosis, bronchiolitis obliterans, severe joint/fascia involvement, and esophageal strictures are the most frequently associated with high morbidity 3 and impaired quality of life. Hence, the big question in the field is how to control cGVHD without increasing the risk of serious adverse effects from immunosuppressive treatment.Cutler et al show that selective inhibition of rho-associated, coiled-coil-containing protein kinase 2 (ROCK2) with belumosudil (formerly known as KD025) is effective and safe in heavily pretreated patients with persistent cGVHD manifestations after 2 to 5 prior systemic lines of therapy (LOTs). The authors are to be congratulated for this trial in these difficult-to-treat patients with advanced stages of cGVHD. Two-thirds of patients had NIH-defined severe cGVHD. Prior treatment included a median of 3 prior LOTs with 27% having at least 5 LOTs. Many of the patients had received extracorporeal photopheresis, ibrutinib, and/or ruxolitinib. Half of patients had 4 or more organs involved, with a high percentage with skin, joints/ fascia, eye, mouth, lung, and/or esophagus involvement. High overall response rates (ORRs) were observed across different organs and prior treatment histories, with partial responses being more frequently recorded in manifestations where fibrosis or permanent organ damage dominated, such as joint/fascia, eyes, skin, or lungs (see figure) . Notably, most patients receiving prior ibrutinib or