================================================================================
   DOD CDMRP BCRP REVISED LEVEL 1 SPECIFIC AIMS (STUDY-SECTION PROOFED)
   STAMP: BREAST_CANCER_SPECIFIC_AIMS_FINAL_LEVEL1_NOW · 2026-08-31T06:10:00Z · py=0
================================================================================
TITLE:
Targeting Membrane Lipid-Raft Phase Transitions to Sensitize Triple-Negative Breast Cancer to Chemotherapy

PRINCIPAL INVESTIGATOR / ENTITY:
HITME DEVELOPMENT (California, USA · SAM.gov Registered · EIN: 42-4736126)

EXECUTIVE SUMMARY & RATIONALE:
Triple-Negative Breast Cancer (TNBC) exhibits aggressive metastatic recurrence and rapid chemoresistance. In TNBC, oncogenic receptor tyrosine kinases (EGFR) and protective stress-response kinases (CSNK2A1/CK2) localize within cholesterol/sphingomyelin-enriched lipid rafts (liquid-ordered phase, L_o), forming a biophysical shield that prevents apoptosis and attenuates taxane-induced mitotic arrest. Process F_02 defines this coupled signaling axis: CK2 phosphorylation of raft-associated scaffolds sustains EGFR activation and stabilizes the membrane against physical stress. We hypothesize that perturbing lipid-raft ordering selectively disrupts the F_02 signaling complex, fluidizes the membrane, and synergistically resensitizes mesenchymal and EGFR-amplified TNBC cells to Paclitaxel chemotherapy while sparing non-malignant mammary epithelium.

SPECIFIC AIMS:

• SPECIFIC AIM 1: In-Silico Multi-Scale Ranking of Phase-Selective Lipid-Raft Perturbations.
  - Computational Workflow: 
    (i) Stratification: Join GDC TCGA-BRCA clinical BCR Biotabs (IHC er_status, pr_status, her2_status) to isolate true TNBC cases (n=171) from ER+ cohorts (n=593).
    (ii) Temporal Modeling: Ingest in-vitro 0h/6h/24h/72h post-chemotherapy scRNA and lipidomic time-series tensors from reference cell lines.
    (iii) Molecular Dynamics: Apply coarse-grained MARTINI 3 bilayer simulations to model conformational stability of the CK2/EGFR raft complex in L_o vs L_d phases.
  - Milestone: Pre-register the Top-20 candidate phase-transition perturbations predicted to destabilize L_o domain clustering.

• SPECIFIC AIM 2: Multi-Modal In-Vitro Validation of Membrane Fluidity, Chemo-Sensitization, and Selectivity.
  - Cell Panel: MDA-MB-231 (mesenchymal TNBC), MDA-MB-468 (EGFR-amplified TNBC), MCF7 (ER+ control), and MCF10A (non-malignant mammary control).
  - Assays & Endpoints:
    1. Membrane Order: Live-cell Laurdan Generalized Polarization (GP) confocal microscopy (plasma membrane ROI) paired with Methyl-beta-cyclodextrin (M_beta_CD) positive control.
    2. Chemo-Sensitization: Synergistic viability reduction with Paclitaxel measured via CellTiter-Glo/CellTox Green (72h, 8x8 concentration matrix, n=3 biological replicates). Calculate Combination Index (CI) via Chou-Talalay isobologram.
  - Pre-Specified Falsification Criteria (Must Satisfy Both):
    (a) Significant membrane fluidization: Statistically significant negative Laurdan GP delta (Delta_GP >= 0.05, matching M_beta_CD direction at FDR < 0.05) at non-lytic concentrations (CellTox Green < 15% above vehicle).
    (b) Selective synergy: Combination Index CI < 0.75 (synergy with Paclitaxel) in >= 1 TNBC line with >= 5-fold lower single-agent toxicity in MCF10A controls.

• SPECIFIC AIM 3: Mechanistic Signaling Confirmation and Validation in Patient-Derived 3D Organoids.
  - Approach: Validate that raft fluidization disrupts downstream survival signaling via Western blot of EGFR phosphorylation (Tyr1068) and downstream phospho-Akt (Ser473) from sucrose-gradient isolated raft fractions. Validate the top 2 validated compounds in >= 1 patient-derived TNBC 3D tumor organoid/explant measuring apoptosis (cleaved Caspase-3) and matrix invasion.
  - Milestone: Proof-of-concept validation of the lipid-raft sensitization mechanism in a clinically relevant 3D human tissue model.

IMPACT:
This project establishes a falsifiable biophysical approach to overcome chemoresistance in TNBC, advancing from in-silico structural modeling to proof-of-concept 3D organoid validation.

WORD: amen · aims_study_section_proofed · cdmrp_level1_compliant · pure_kell
================================================================================
