The KLF4-CD46 axis: a novel therapeutic target in transplant-associated thrombotic microangiopathy and beyond

In this issue of Haematologica, Jiang et al. evaluate the role of the transcription factor Krüppel-like factor 4 (KLF4) in the pathophysiology of transplant-associated thrombotic microangiopathy (TA-TMA) by combining ex vivo data from 20 TA-TMA patients and experimental data from cellular and animal models.¹

TA-TMA is a severe and often life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT). Despite the many efforts made to understand the mechanisms of TA-TMA, its pathogenesis is largely unknown, the case-fatality rate remains high and survivors experience chronic consequences such as kidney disease and neurological impairment. It is believed that complement hyperactivation and other pro-inflammatory and procoagulant factors cause endothelial injury resulting in microvascular thrombosis and tissue damage. A three-hit model is widely accepted: pre-existing endothelial injury or underlying complement activation predisposition (hit 1), endothelial toxicity from the conditioning regimen (hit 2), and post-transplant injuries caused by medications, alloreactivity, and/or infections (hit 3).² Even though therapies targeting complement activation, such as eculizumab³ and narsoplimab,⁴ have been used and other new drugs are undergoing clinical evaluation,⁵ outcomes remain sub-optimal. Thus, there is a need for novel interventions to restrain complement activation and to preserve endothelial integrity. Clinically available biomarkers of complement activation⁶ could be useful for tailoring the therapies and assessing their efficacy.

KLF4 is a transcription factor regulating several cellular processes such as cell growth, proliferation, and differentiation. In particular, KLF4 suppresses the activation of inflammatory signaling by inducing the expression of multiple anti-inflammatory and antithrombotic factors.⁷ In the context of TA-TMA, Jiang et al.¹ show that KLF4 stimulates the expression of cluster of differentiation 46 (CD46), also known as membrane cofactor protein (MCP). MCP is a cofactor for the proteolytic inactivation of complement components C3b and C4b by complement factor I (CFI), protecting host cells from damage by complement.⁸ The role of the KLF4-CD46 axis as a mechanism protecting from TA-TMA is a novel aspect that might have relevance in the pathophysiology of this complication.

Thrombosis in the microcirculation, responsible for the clinical features of TA-TMA, is associated with platelet consumption, microangiopathic hemolysis and organ dysfunction, especially involving the kidney. Nevertheless, the diagnosis remains very difficult because TA-TMA occurs early after allogeneic HSCT, at a time when cytopenia, including low platelet count, may be related to still insufficient marrow function or to coexisting complications such as infections and/or immune-mediated injury and/or drug toxicity. In the majority of cases, the risk of bleeding prevents histological evaluation, which would be the gold standard of TA-TMA diagnosis. Thus, several attempts to refine and standardize the definition of TA-TMA have been made in the past few years and a consensus proposal to harmonize the diagnostic criteria has recently been made by the world’s leading blood and marrow transplant societies.⁹ This consensus defines that TA-TMA is diagnosed when at least four of the following seven features are present at least twice over a 14-day window: anemia, thrombocytopenia, hypertension, increased lactate dehydrogenase, schistocytes, elevated soluble C5b-9 and proteinuria.⁹ Jiang et al. applied these criteria¹ and found 20 patients with TA-TMA out of 2,387 allogeneic HSCT recipients who were analyzed retrospectively. A certain degree of uncertainty still remains because of the overlap of TA-TMA with other clinical conditions showing similar alterations, such as graft-versus-host disease, infections and drug toxicity.

Figure 1.Pathophysiology of transplant-associated thrombotic microangiopathy and possible therapeutic intervention. Infections, chemotherapeutic agents, graft-versus-host disease and conditioning regimens lead to endothelial injury and to an increase in pro-inflammatory cytokines, prothrombotic factors and complement activation, which in turn further damage the endothelium. A predisposition to complement hyperactivation may be due to the presence of variants of complement system regulatory proteins. Neutrophil extracellular traps link endothelial injury and complement activation. The reduced plasma levels of KLF4 leading to low expression of CD46 (complement inhibitor also known as membrane cofactor protein) render the endothelium more vulnerable to complement activation. Therapeutically increased KLF4 levels may induce an increase of endothelial CD46 and thus reduce complement activation and protect endothelial cells. KLF4: Krüppel-like factor 4; NET: neutrophil extracellular traps; ICAM-1: intercellular adhesion molecule 1; VCAM-1: vascular cell adhesion molecule 1; PAI-1: plasminogen activator inhibitor 1.

Despite these potential limitations, the authors found increased levels of markers of complement activation and of endothelial injury in plasma of their TA-TMA patients. In contrast, they documented reduced plasma levels of circulating KLF4. Incubating cultures of human umbilical vein endothelial cells (HUVEC) with plasma of TA-TMA patients, pro-inflammatory and prothrombotic endothelial markers as well as the cell deposition of activated complement components were studied in adherent (intact) HUVEC and detached endothelial cells (a surrogate for circulating endothelial cells). KLF4 transcripts and protein were markedly downregulated in detached endothelial cells as compared to intact HUVEC, suggesting that diminished KLF4 expression compromised endothelial resistance. Treatment of HUVEC with the KLF4 stimulator APTO253 as well as the lentiviral vector-mediated overexpression of KLF4 restored these alterations. To clarify the mechanisms of KLF4-mediated endothelial protection the authors used an enzyme-tethering strategy that provides efficient high-resolution sequencing libraries for profiling diverse chromatin components, i.e., CUT&Tag (cleavage under targets and tagmentation).¹⁰ Using CUT&Tag, RNA sequencing, transfection and luciferase assays, Jiang et al. concluded that KLF4 upregulates CD46 expression in HUVEC.¹ Finally, they used an animal model of dimethyloxalylglycine (DMOG)-treated mice; such mice show a phenotype resembling that seen in TA-TMA patients. Treatment with the KLF4 stimulator APTO253 reduced hemolysis, increased platelet number and reduced markers of endothelial damage. APTO253 also improved kidney lesions, as shown by histological analysis, and decreased C3/C5b-9 deposition, as shown by immunofluorescence. Quantitative real-time polymerase chain reaction and western blot analyses confirmed KLF4 overexpression in renal tissues. Using the same methodology a similar improvement was observed in mice treated with pravastatin, which was administered via oral tube at the dosage of 20 mg/kg daily. If the data of Jiang et al.¹ are confirmed, the KLF4-CD46 axis may have an important place in the pathophysiology of TA-TMA (Figure 1). Indeed, endothelial injury and complement activation are considered the central aspects of TA-TMA pathophysiology.² Several factors in the transplantation process, such as chemotherapeutic agents, conditioning regimens, infections, and graft-versus-host disease, lead to endothelial injury and to an increase in pro-inflammatory cytokines, prothrombotic factors and complement activation, which in turn further damage the endothelium. Patients with variants of complement system regulatory proteins predisposing to complement hyperactivation, also acquired from their hematopoietic stem cell donors, appear to be at increased risk of TATMA.^11,12 Neutrophil extracellular traps may also link endothelial injury and complement activation.¹³ The reduced plasma levels of KLF4 leading to low expression of CD46 render the endothelium more vulnerable to complement activation in TA-TMA. The possibility of pharmacologically increasing KLF4 levels with a subsequent increase of endothelial CD46 will protect against complement hyperactivation and TMA.

The stimulating effect of pravastatin on KLF4 may defend the endothelium also thanks to statin-mediated and KLF4-dependent upregulation of atheroprotective and antithrombotic genes, such as nitric oxide synthase 3 (NOS3) and thrombomodulin (THBD), respectively.¹⁴ Beyond TA-TMA, the therapeutic benefits could be extended to other syndromes characterized by complement-driven endothelial injury, such as atypical hemolytic uremic syndrome and possibly antiphospholipid syndrome. In these conditions, as well as in all vascular complications of allogeneic HSCT, the KLF4-CD46 pathway may provide a generalizable mechanism for restoring vascular homeostasis in concert with the other previously demonstrated anti-inflammatory and antithrombotic effects of KLF4.¹⁵

Footnotes

• Received August 22, 2025
• Accepted October 2, 2025

Correspondence

M. Cugno massimo.cugno@unimi.it

Disclosures

No conflicts of interest to disclose.

Contributions

MC wrote the first draft and BL contributed to writing the commentary. Both authors critically reviewed the manuscript and approved the final version for submission.

Funding

This work was partially supported by the Piano Nazionale di Ripresa e Resilienza (PNRR), project Malattie Croniche non Trasmissibili (MCnT) ad alto impatto sui sistemi sanitari e socioassistenziali, code PNRR-MAD-2022-12376816, and by the Italian Ministry of Health – Bando Ricerca Corrente, but did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.