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Employing CRISPR-Cas9 to Generate CD133 Synthetic Lethal Melanoma Stem Cells
Cynthia M Simbulan-Rosenthal1, Yogameenakshi Haribabu1, Sahar Vakili1
1Department of Biochemistry and Molecular & Cellular Biology, Georgetown University School of Medicine, Washington, DC 20057, USA.
Abstract:
Malignant melanoma is a lethal skin cancer containing melanoma-initiating cells (MIC) implicated in tumorigenesis, invasion, and drug resistance, and is characterized by the elevated expression of stem cell markers, including CD133. The siRNA knockdown of CD133 enhances apoptosis induced by the MEK inhibitor trametinib in melanoma cells. This study investigates the underlying mechanisms of CD133's anti-apoptotic activity in patient-derived BAKP and POT cells, harboring difficult-to-treat NRASQ61K and NRASQ61R drivers, after CRISPR-Cas9 CD133 knockout or Dox-inducible expression of CD133. MACS-sorted CD133(+) BAKP cells were conditionally reprogrammed to derive BAKR cells with sustained CD133 expression and MIC features. Compared to BAKP, CD133(+) BAKR exhibit increased cell survival and reduced apoptosis in response to trametinib or the chemotherapeutic dacarbazine (DTIC). CRISPR-Cas9-mediated CD133 knockout in BAKR cells (BAKR-KO) re-sensitized cells to trametinib. CD133 knockout in BAKP and POT cells increased trametinib-induced apoptosis by reducing anti-apoptotic BCL-xL, p-AKT, and p-BAD and increasing pro-apoptotic BAX. Conversely, Dox-induced CD133 expression diminished apoptosis in both trametinib-treated cell lines, coincident with elevated p-AKT, p-BAD, BCL-2, and BCL-xL and decreased activation of BAX and caspases-3 and -9. AKT1/2 siRNA knockdown or inhibition of BCL-2 family members with navitoclax (ABT-263) in BAKP-KO cells further enhanced caspase-mediated apoptotic PARP cleavage. CD133 may therefore activate a survival pathway where (1) increased AKT phosphorylation and activation induces (2) BAD phosphorylation and inactivation, (3) decreases BAX activation, and (4) reduces caspases-3 and -9 activity and caspase-mediated PARP cleavage, leading to apoptosis suppression and drug resistance in melanoma. Targeting nodes of the CD133, AKT, or BCL-2 survival pathways with trametinib highlights the potential for combination therapies for NRAS-mutant melanoma stem cells for the development of more effective treatments for patients with high-risk melanoma.
Insights
CD133 (a stem cell marker) promotes melanoma cell survival and drug resistance by activating the AKT/BAD pathway. Targeting CD133, AKT, or BCL-2 enhances apoptosis in NRAS-mutant melanoma stem cells, offering new therapeutic strategies.
Area of Science:
- Oncology
- Molecular Biology
- Dermatology
Background:
- Malignant melanoma, a lethal skin cancer, is driven by melanoma-initiating cells (MICs) expressing stem cell markers like CD133.
- Elevated CD133 expression is linked to tumorigenesis, invasion, and drug resistance in melanoma.
- NRAS-mutant melanomas, particularly those with Q61K/R mutations, present treatment challenges.
Purpose of the Study:
- To investigate the mechanisms by which CD133 confers anti-apoptotic activity in NRAS-mutant melanoma cells.
- To determine the role of CD133 in regulating survival pathways, including AKT and BCL-2 family members.
- To evaluate the therapeutic potential of targeting CD133 in combination with MEK inhibitors like trametinib.
Main Methods:
- CRISPR-Cas9 gene editing to knockout CD133 in patient-derived melanoma cells (BAKP, POT, BAKR).
- Doxycycline-inducible expression of CD133 to study its functional effects.
- Cellular assays to assess apoptosis, cell survival, and protein activation (AKT, BAD, BAX, caspases).
- siRNA knockdown and pharmacological inhibition of key pathway components (AKT, BCL-2 family).
Main Results:
- CD133 knockout sensitized melanoma cells to trametinib and dacarbazine by increasing apoptosis.
- CD133 knockout reduced anti-apoptotic proteins (BCL-xL, p-AKT, p-BAD) and increased pro-apoptotic BAX.
- CD133 expression promoted cell survival and drug resistance, associated with elevated p-AKT, p-BAD, BCL-2, BCL-xL, and reduced BAX and caspase activation.
- Targeting AKT or BCL-2 family members further enhanced apoptosis in CD133-knockout cells.
Conclusions:
- CD133 activates an AKT-dependent survival pathway that suppresses apoptosis and confers drug resistance in melanoma.
- This pathway involves BAD phosphorylation, reduced BAX activation, and decreased caspase activity.
- Targeting CD133, AKT, or BCL-2 pathways offers a promising strategy for combination therapies against NRAS-mutant melanoma stem cells.
