How understanding the complex MARCKS expression could help cure rare diseases

The National Organization for Rare Disorders at NC State just celebrated its first virtual Rare disease day on February 25 2021 in collaboration with the University of North Carolina at Chapel Hill and Wake Forest Medical School. Our research mentor, Dr. Kenneth Adler represented our chapter as our keynote speaker.

Dr. Adler is a professor of Cell Biology at NC State’s College of Veterinary medicine and he believes medical research has benefitted greatly from increased interest.

“Advocacy is playing an increasingly important role in accelerating progress in understanding and treating rare diseases,” Dr. Adler said. “Rare diseases of all kinds require dedicated individuals to pursue treatments and cures.”

Both at the University and at his Biomarck Pharamceuticals start-up, Dr. Adler’s work focuses on the so-called MARCKS expression and ways to counter it.

The Myristoylated Alanine Rich C-Kinase Substrate (MARCKS) is encoded by the MARCKS gene in vertebrates, a gene that plays an important role in many cellular processes, including cell motility, secretion, regulation of the cell cycle, and neural development.

Significantly heightened levels of MARCKS expression are found in many cell primary tumor samples, especially in lung & breast cancers. However, MARCKS expression is not accompanied by a mutation, leading to an understanding that the MARCKS pathway is a contributor to the cancer phenotype.

Step 1: Phosphorylation of MARCKS protein

It all starts with the so-called ATP, Adenosine Triphosphate by its full name. ATP is a molecule that captures chemical energy from the breakdown of food molecules to fuel bodily processes. Some enzymes (called kinases) can take phosphate (PO4) from ATP and bring them to other molecules in the body (called a substrate) — this process is called phosphorylation.

A first step towards understanding the MARCKS mechanism is breaking down the termprotein-kinase phosphorylation, which is depicted in thet image below:

A protein kinase catalyzes the transfer of phosphate from ATP to its protein substrates. Protein kinases are divided into many families, but the one protein kinase classification relevant to understanding the MARCKS mechanism is the Serine/ Threonine protein kinase.

This particular family of proteins includes cyclins or proteins associated with cell division, as well as Protein kinase D, which is increasingly recognized as a key regulatory signaling hub.

• CDKs combine with a cyclin to regulate different phases of the cell cycle from the G1 phase to the completion of the cell cycle. G1 is the growth phase of the cell cycle before the DNA is synthesized and when the most proteins are made.
• Protein Kinase D is activated by protein kinase C and is involved in the regulation of cell growth, proliferation, cell migration (metastasis), differentiation, and apoptosis (natural cell death). Protein Kinase C (PKC) is a family of protein kinase enzymes that are involved in controlling the function of other proteins.

MARCKS is a substrate for G1 kinases (CDK 4/6) as well as for the protein kinase C. Signal transduction pathways are cellular responses to extracellular stimuli, for Kinase C the signal transduction cascades include cell proliferation and immunological responses. Thus, MARCKS seems to be the link between cell cycle signaling and Protein Kinase C signal transduction pathways. This further indicates an involvement of the MARCKS proteins in the cancer phenotype.

In the above schematic of the MARCKS substrate, each region corresponds to a particular function:

1. N-terminal – Start of a protein, in the above picture Mystrisic acid (MA) is linked to the N terminal of the protein
2. MH2- The function of the MH2 domain is currently unknown. (Sheats et. al)
3. Phosphorylation site domain (PSD)- site of phosphorylation, MARCKS binds to the cell membrane in its dephosphorylated state by ionic interactions between PSD site and membrane phospholipids

Step 2: PIP2 Release from membrane sites

MARCKS is usually bound to a lipid messenger called PIP2. When MARCKS is phosphorylated, (PIP2 – MARCKS) binding is disrupted, and PIP2 is released from its sequestration sites in the membrane, allowing detachment. This activates other signaling molecules such as Focal Adhesion Kinase (FAK) and other cytoskeletal proteins. In cancer, this results in enhanced cell migration (metastasis) and proliferation.

Step 3: FAK Activation

FAK is a key regulator of the growth factor receptor. Increased FAK expression is observed in many cancer cells, high expression is associated with poor chances of recovery. The first step in FAK activation is binding with PIP2. FAK promotes malignancy via highly coordinated signaling events that use a diverse range of cellular processes, especially cell migration (metastasis) and invasion.

BIO-11006 (anti-MARCKS peptide)

Biomarck Pharmaceuticals is currently developing its lead compound, BIO-11006 for non-small cell lung cancer and acute respiratory distress syndrome (ARDS). This is a 10 amino acid sequence attached at the N-terminal of MARCKS Protein. It is an investigational peptide medication delivered by a nebulizer which has shown activity in the laboratory and in animal studies in certain types of cancer.

It has been granted FDA acceptance to begin studies in adult lung cancer. BIO-11006 functions by disrupting the very first step of phosphorylating MARCKS and by selectively dephosphorylating MARCKS, inhibiting all the consecutive steps.

Current Use of BIO-110066 in Rare pediatric cancers

Compassionate use encompasses expanded access for a patient with an immediately life threatening condition. They gain access to an investigational drug for treatment outside the clinical trials when noo comparable or satisfactory alternate therapy is available.

In 2016, CBS North Carolina reported the first instance of compassionate use of BIO-110066. Philomena Stendardo, 8, was diagnosed with Diffuse Intrinsic Pontine Glioma (DIPG). DIPG is a rare, aggressive brain tumor. The tumor wrapped around her brain stem was inoperable. Biomarck Pharmaceuticals partnered with the Live Like Bella foundation to implement compassionate use in this case. After a few weeks of being administered with 006, Stendardo’s condition improved, she was able to sit up on her own, walk with assistance, move her right side and was in a position to think more clearly. Unfortunately she passed away several months, but her treatment could be a stepping stone to improving the quality of life for a cancer patient.

“The initial results suggest that there is something in this treatment that could be beneficial to patients with rare pediatric cancers,” Dr. Adler said, “Currently we are conducting expanded access clinical trials in Osteosarcoma patients in collaboration with Nicklaus Children’s hospital in Miami.”

References:

1. (1) Rare Disease day 2021- NORD at NC State University, UNC-Ch, Wake forest Med and Tompkins HS – YouTube
2. Biomarck.com 
3. (1) BMK101 MOA Final 180914e – YouTube
4. Kinase Classification-CUSABIO
5. MOA-Adler.pptx (biomarck.com)
6. Child with rare cancer is first to receive NC State scientist’s experimental treatment (wbtv.com)
7. Sheats, M. K., Yin, Q., Fang, S., Park, J., Crews, A. L., Parikh, I., Dickson, B., & Adler, K. B. (2019). MARCKS and Lung Disease. American journal of respiratory cell and molecular biology, 60(1), 16–27. https://doi.org/10.1165/rcmb.2018-0285TR
8. Chen, C. H., Statt, S., Chiu, C. L., Thai, P., Arif, M., Adler, K. B., & Wu, R. (2014). Targeting myristoylated alanine-rich C kinase substrate phosphorylation site domain in lung cancer. Mechanisms and therapeutic implications. American journal of respiratory and critical care medicine, 190(10), 1127–1138. https://doi.org/10.1164/rccm.201408-1505OC
9. Yin Q, Fang S, Park J, Crews AL, Parikh I, Adler KB. An Inhaled Inhibitor of Myristoylated Alanine-Rich C Kinase Substrate Reverses LPS-Induced Acute Lung Injury in Mice. Am J Respir Cell Mol Biol. 2016 Nov;55(5):617-622. doi: 10.1165/rcmb.2016-0236RC. PMID: 27556883; PMCID: PMC5105187.
10. Gambhir A, Hangyás-Mihályné G, Zaitseva I, Cafiso DS, Wang J, Murray D, Pentyala SN, Smith SO, McLaughlin S. Electrostatic sequestration of PIP2 on phospholipid membranes by basic/aromatic regions of proteins. Biophys J. 2004 Apr;86(4):2188-207. doi: 10.1016/S0006-3495(04)74278-2. PMID: 15041659; PMCID: PMC1304070.
11. Chen, CH., Fong, L., Yu, E. et al. Upregulation of MARCKS in kidney cancer and its potential as a therapeutic target. Oncogene 36, 3588–3598 (2017). https://doi.org/10.1038/onc.2016.510
12. Chen CH, Thai P, Yoneda K, Adler KB, Yang PC, Wu R. A peptide that inhibits function of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) reduces lung cancer metastasis. Oncogene. 2014 Jul 10;33(28):3696-706. doi: 10.1038/onc.2013.336. Epub 2013 Aug 19. PMID: 23955080; PMCID: PMC4631387.
13. Yin Q, Fang S, Park J, Crews A, Parikh I, Dickson B et al., Marcks- inhibitory peptides synergize with Cisplatin to inhibit metastasis and primary tumor growth in mouse orthotopic lung cancer models, Am J Respir, Crit CARE MED 2016, 193: A3131