FLT4 Fc Human

What is the structural composition of the FLT4 receptor?

FLT4 is a class III receptor tyrosine kinase characterized by seven immunoglobulin-like loops in its extracellular domain, containing 12 potential glycosylation sites. It belongs to a subfamily that includes FLT1 and KDR/FLK1 (kinase insert domain-containing receptor/fetal liver kinase 1) receptors. The full-length sequence reveals distinctive structural elements that contribute to its specialized function in lymphatic development . The receptor contains important functional domains including an extracellular ligand-binding region, a transmembrane domain, and an intracellular tyrosine kinase domain that mediates downstream signaling.

What expression patterns does FLT4 demonstrate in normal tissue?

FLT4 is expressed as 5.8- and 4.5-kilobase mRNAs with differences in their 3' sequences, showing differential expression patterns across cell types. In HEL and DAMI leukemia cells, these transcript variants display distinct expression profiles . Most fetal tissues express FLT4 mRNA, with highest levels detected in spleen, brain intermediate zone, and lung. In situ hybridization studies have shown FLT4 autoradiographic grains decorating bronchial epithelial cells of fetal lung, while early studies found no evidence for FLT4 expression in endothelial cells of blood vessels . This tissue-specific expression pattern suggests specialized roles in organ development.

What is the primary signaling function of FLT4?

FLT4 serves as a cell-surface tyrosine kinase receptor for vascular endothelial growth factors C and D (VEGFC and VEGFD). When these ligands bind to FLT4/VEGFR3, downstream signaling docking sites are produced, regulating the proliferation, migration, and survival of lymphatic endothelial cells (LECs) . This signaling cascade is essential for proper lymphatic system development and maintenance. Disruption of this pathway, as seen in the Chy mouse model with a heterozygous FLT4 variant in the tyrosine kinase domain, prevents proper phosphorylation and results in early developmental deficiencies in some lymphatic vessels .

How can researchers effectively target FLT4 in experimental models?

Several approaches have been developed to modulate FLT4 activity experimentally:

Peptide-based inhibition: Researchers have designed targeted peptides that can disrupt FLT4 signaling. In one study, 12 peptides targeting FLT4 were developed, with 4 selected for further examination. Peptide P4, which targets the intracellular domain of FLT4, effectively increased IFN-γ by inhibiting FLT4 activation . This approach involved:

• Designing peptides not exceeding 18-mers based on sequences in the intracellular region

• Testing at concentrations of 25 μM in vitro and 20 mg/kg in vivo

• Evaluating effects on immune cell populations using flow cytometry

• Measuring downstream effects including IFN-γ production

Small molecule inhibition: Small molecule inhibitors like MAZ51 have been used to block FLT4 activity, effectively reducing leukemic cell-derived colony-forming units and increasing apoptosis when co-treated with cytosine arabinoside under vascular endothelial growth factor C stimulation .

What experimental methods are optimal for studying FLT4 variants in lymphatic disorders?

Research methodologies for studying FLT4 variants include:

Genetic sequencing approaches: Trio-whole-exome sequencing (Trio-WES) has been effectively used to analyze underlying genetic causes of primary lymphedema. Sanger sequencing validation helps confirm variants in affected probands and unaffected family members .

In silico analysis tools: Multiple computational methods predict the pathogenicity of FLT4 variants:

• CADD, SIFT, DANN, MetaSVM, MetaLR, and M-CAP for missense variants

• Multiple sequence alignment to determine evolutionary conservation

• 3D modeling using tools like SWISS-MODEL to visualize structural changes

Mouse models: The Chy mouse possessing a heterozygous FLT4 variant in the tyrosine kinase domain serves as a valuable model for studying lymphatic vessel deficiencies .

How should researchers interpret discrepancies in FLT4 expression data between studies?

When reconciling contradictory findings regarding FLT4 expression:

• Consider developmental context: Expression patterns may vary significantly between embryonic, fetal, and adult tissues

• Evaluate methodology differences: Results from in situ hybridization, immunohistochemistry, and RNA sequencing may yield different findings

• Examine cell-specific resolution: Single-cell approaches may reveal heterogeneity masked in bulk tissue analyses

• Account for pathological states: Disease conditions may alter normal expression patterns

Studies have yielded apparently contradictory results, with some early research finding no evidence for FLT4 expression in blood vessel endothelial cells , while others demonstrate expression in specific vascular beds under certain conditions.

What methodologies can effectively assess FLT4's role in acute myeloid leukemia (AML)?

Researchers investigating FLT4 in AML should consider these approaches:

Animal models: Studies have demonstrated that inhibition or absence of FLT4 in AML blasts suppresses homing to bone marrow in immunocompromised mice and blocks engraftment of AML blasts .

Ex vivo culture systems: Examining the effects of FLT4 inhibition on patient-derived samples:

• Colony formation assays to assess clonogenic potential

• Apoptosis measurements following treatment with FLT4 inhibitors

• Combination treatment approaches with standard chemotherapeutics

Flow cytometry analysis: Evaluating FLT4 expression in different cellular compartments and correlating with clinical outcomes. High cytosolic FLT4 expression has been linked to AML-refractory status through an internalization mechanism .

How can FLT4-targeting approaches be integrated into immunotherapy strategies?

FLT4-targeting peptides have shown potential for modulating the tumor immune microenvironment:

• Restoration of dysfunctional AML-NK cells through FLT4 inhibition

• Increased IFN-γ expression in both NK cells and T cells

• Reduction in immunosuppressive regulatory T cell frequencies

• Synergistic effects when combined with conventional chemotherapy like cytosine arabinoside

These approaches demonstrate that FLT4-targeting strategies not only affect the malignant cells directly but also modulate the immune response to create a more favorable anti-tumor environment.

What predictive value does FLT4 expression have for treatment resistance in AML?

High expression of FLT4 is associated with increased cancer activity in AML and has biological functions in both leukemogenesis and refractoriness . When establishing FLT4 as a prognostic marker:

• Quantify FLT4 expression levels in patient samples using standardized methods

• Correlate expression with treatment response and survival outcomes

• Determine cellular localization (membrane vs. cytosolic) of FLT4, as high cytosolic FLT4 is linked to refractory disease

• Evaluate the effect of FLT4 inhibition on chemotherapy sensitivity in patient-derived samples

This approach provides a framework for stratifying patients and potentially guiding treatment decisions based on FLT4 expression profiles.

How should researchers investigate novel FLT4 variants identified in clinical cases?

A systematic approach includes:

• Genetic characterization: Document the precise variant using standard nomenclature and determine inheritance patterns

• Functional validation: Assess the impact on protein structure, ligand binding, and downstream signaling

• Phenotype correlation: Compare clinical manifestations with previously reported cases

• In silico analysis: Use computational tools to predict pathogenicity

A case study example demonstrates this approach with compound heterozygotes for the FLT4 gene carrying two different variants - a missense variant (NM_182925.5; c.1504G>A, p.Glu502Lys) and a recurrent deletion variant (NM_182925.5; c.3323_3325del, p.Phe1108del) . In silico analysis revealed:

• The missense variant results in replacement of glutamic acid (acidic) with lysine (basic)

• Multiple predictive tools classified this variant as disease-causing

• Multiple sequence alignment confirmed evolutionary conservation of affected residues

• 3D modeling showed changes in polarity for the missense variant and destruction of α-helix structure for the deletion variant

What prenatal diagnostic approaches exist for FLT4-related disorders?

Prenatal detection of FLT4-related disorders involves:

• Ultrasonography: May reveal hydrops fetalis, increased nuchal translucency, or hydrothorax

• Genetic testing: Exome sequencing can identify causative FLT4 variations

• Comparative genomic hybridization: Rules out large chromosomal abnormalities

Case studies report variations such as p.(Ser1275Thr) and p.(Ser1275Arg) in fetuses presenting with congenital lymphedema . The identification of de novo mutations at the same codon in multiple cases suggests possible genotype-phenotype correlations that warrant further investigation.

How does inheritance pattern influence the phenotypic expression of FLT4 variants?

FLT4-related disorders demonstrate complex inheritance patterns:

• Autosomal dominant: Most commonly reported with incomplete penetrance

• Autosomal recessive: Reported in cases with compound heterozygous variants

In a Chinese family study, unaffected parents carried single heterozygous variants, while the affected proband inherited both variants (compound heterozygote) . This suggests that in some cases, two variants may be necessary for phenotypic expression, highlighting the importance of complete family studies when investigating suspected FLT4-related disorders.

What design considerations are critical for developing FLT4-targeting peptides?

When developing peptides targeting FLT4:

• Target selection: Focus on functionally critical domains such as the intracellular kinase region

• Size optimization: Peptides not exceeding 18-mers have shown efficacy

• Binding specificity: Target regions like Thr444, Thr446, Glu426, and Lys516 in the Fc domain of FLT4 that are essential for activation

• Structural disruption: Design peptides that disrupt homotypic interactions in D5 of the Fc domain

Research indicates that the signaling domain of FLT4 is susceptible to disruption by homodimers, making FLT4 activation vulnerable to inhibition by appropriately designed peptides .

How can researchers evaluate the efficacy of FLT4-targeting therapeutics in preclinical models?

A comprehensive assessment includes:

• In vitro binding studies: Confirm target engagement

• Cellular assays: Measure effects on downstream signaling events

• Ex vivo testing: Evaluate impact on patient-derived samples

• Animal models: Assess in vivo efficacy using appropriate disease models

In AML studies, researchers have demonstrated that FLT4-targeting peptides can:

• Reduce colony formation by leukemic cells

• Increase apoptosis when combined with standard chemotherapy

• Restore NK cell function in a leukemic environment

• Decrease regulatory T cell frequency

• Increase IFN-γ production by effector immune cells

These multifaceted assessments provide a more complete picture of therapeutic potential than single-endpoint studies.

How might FLT4-targeting approaches be integrated with existing treatment paradigms?

Researchers should consider:

• Combination approaches: FLT4 inhibitors have shown synergistic effects with conventional chemotherapeutics like cytosine arabinoside in AML models

• Sequential therapy strategies: Determining optimal timing of FLT4 targeting relative to other treatments

• Biomarker-guided patient selection: Using FLT4 expression levels to identify patients most likely to benefit

• Immune-modulating effects: Leveraging the immunomodulatory properties of FLT4 inhibition alongside direct anti-tumor effects

The dual action of FLT4-targeting therapeutics—directly affecting malignant cells while also modulating the immune environment—opens opportunities for novel combination approaches in conditions like refractory AML .