Brooke Keeton and Dr. John Prince, Department of Chemistry and Biochemistry

B-cell chronic lymphocytic leukemia (B-CLL) is the most common type of leukemia in adults.1 Over 75% of patients diagnosed with B-CLL are over the age of 50 years old, and have a median survival between 18 months and 3 years.2 B-CLL originates from a mutation in the DNA of white blood cells, the potent infection fighters of the body. This genetic mutation produces abnormal white blood cells, rendering them nonfunctional and resulting in a weak immune system. While this mutation originally occurs in the bone marrow, it slowly invades other parts of the body, including the lymph nodes, liver, and spleen.

Mutations that give rise to B-CLL commonly affect the cell’s apoptosis cycle (programmed cell death), rather than increased cell proliferation (cell division). Common drug treatments for leukemia take advantage of the increased cell proliferation characteristic. Unfortunately, because B-CLL does not experience increased cell division, these traditional forms of drug treatment do not work. The demand for a B-CLL treatment has driven researchers to test a variety of drugs. The most promising is flavopiridol.

Flavopiridol is a drug developed by our collaborator, David Bearss (P.D. Bio Dept.). Clinical results have shown that flavopiridol treatment of B-CLL patients can cause different responses: 30% of the patients lack response, 40% of the patients were cured without remission, whereas another 30% of the patients died of tumor lysis syndrome. Being able to predict how a patient will respond to flavopiridol treatment will allow for flavopirodol treatment on a large subset of the B-CLL patient population.

B-CLL drug resistance to common drug treatment is primarily caused by Bcl-2 and Mcl-1, two proteins of the mitochondrial outer membrane (OMM). The anti-cancer drug flavopiridol targets the positive transcription elongation factor (P-TEFb), which leads to inhibition of P-TEFb and the loss of mRNA production3. In B-CLL, Bcl-2 and Mcl-1 sustain a malignant phenotype, which exhibit short half-lives in their mRNA and protein. Thus, Bcl-2 and Mcl-1 can be the most likely therapeutic targets of transcriptional 1 Byrd, John. “Chronic Lymphocytic Leukemia”. Leukemia & Lymphoma Society. Retrieved 24 March 2014 2 Byrd, John. “Randomized phase 2 study of fludarabine with concurrent versus sequential treatment with rituximab in symptomatic, untreated patients with B-cell chronic lymphocytic leukemia: results from Cancer and Leukemia Group B 9712 (CALGB 9712)” 3 Chen, R.; Keating, M. J.; Gandhi, V.; Plunkett, W., Transcription inhibition by flavopiridol: mechanism of chronic lymphocytic leukemia cell death. Blood 2005, 106 (7), 2513-9. inhibitors flavopiridol. Bcl-2 and Mcl-1 both exert the anti-apoptosis activity on the OMM, where they can antagonize BAX and BAK activation to maintain mitochondrial integrity4. Decreased Bcl-2 and Mcl-1 should release activation of BAX and BAK, which can induce mitochondrial outer membrane permeability (MOMP) by interaction with OMM. However, treatment with flavopiridol on B-CLL patients has led to different responses. A variety of proteins in the outer mitochondrial membrane could be altering the response of patients to flavopiridol. Alternatively, lipid constituents are known to strongly influence the behavior of membrane proteins. For instance, Sphingolipids, particularly sphingosin-1-PO4 and hexadecenal, have been previously shown to cooperate with BAK and BAX respectively to promote the mitochondrial pathway of apoptosis5. We are, therefore, testing the hypothesis that the abundance of different mitochondrial lipids, or the expression of different mitochondrial proteins, will be predictive of response to flavopiridol treatment. Data gathered may also lead to a more complete understanding of OMM protein and lipid regulators.

The white blood cells of B-CLL patients before treatment with flavopiridol along with age, gender, and their post-treatment response were studied. The total mitochondrial lipid and protein content in these samples were then mesasured using a high mass accuracy LTQ-Orbitrap XL mass spectrometer. The data was then put through a machine learning procedure to determine the predictive value of the lipid/protein profiles. Originally, the mitochondria fraction from lymphocytes were isolated as described by Wieckowski et al.6 and purity tested by Western blot against the OMM protein VDAC-1. We then attempted lipid extraction with the Folch method, ionized with electrospray ionization (ESI) in both positive and negative mode and analyzed with mass spectrometry (MS).

Lipid quantities and identities are determined with in-house software using the LipidMaps database. Proteins were then processed with Filter-Aided Sample Preparation (FASP) method as described by Mann’s group7. Digested proteins were then separated by Ultra High Performance Liquid Chromatography, before MS analysis, and proteins identified by Mascot database search. Classification (i.e., machine learning) using lipid/protein profiles were performed using one-against-one multiclass support vector machines. Unfortunately, our original extraction method struggled to isolate the mitochondria fraction, skewing our results. Current methods are showing promise, but the greatest obstacle is still finding an effective extraction method. Research is still being conducted to improve extraction methods.

1 Byrd, John. “Chronic Lymphocytic Leukemia”. Leukemia & Lymphoma Society. Retrieved 24 March 2014 2 Byrd, John. “Randomized phase 2 study of fludarabine with concurrent versus sequential treatment with rituximab in symptomatic, untreated patients with B-cell chronic lymphocytic leukemia: results from Cancer and Leukemia Group B 9712 (CALGB 9712)” 3 Chen, R.; Keating, M. J.; Gandhi, V.; Plunkett, W., Transcription inhibition by flavopiridol: mechanism of chronic lymphocytic leukemia cell death. Blood 2005, 106 (7), 2513-9. 4 Perciavalle, R. M.; Stewart, D. P.; Koss, B.; Lynch, J.; Milasta, S.; Bathina, M.; Temirov, J.; Cleland, M. M.; Pelletier, S.; Schuetz, J. D.; Youle, R. J.; Green, D. R.; Opferman, J. T., Anti-apoptotic MCL-1 localizes to the mitochondrial matrix and couples mitochondrial fusion to respiration. Nature cell biology 2012, 14 (6), 575-83 5 Chipuk, J. E.; McStay, G. P.; Bharti, A.; Kuwana, T.; Clarke, C. J.; Siskind, L. J.; Obeid, L. M.; Green, D. R., Sphingolipid metabolism cooperates with BAK and BAX to promote the mitochondrial pathway of apoptosis. Cell 2012, 148 (5), 988-1000. 6 Wieckowski, M. R.; Giorgi, C.; Lebiedzinska, M.; Duszynski, J.; Pinton, P., Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells. Nature protocols 2009, 4 (11), 1582-90. 7 Wisniewski, J. R.; Zougman, A.; Nagaraj, N.; Mann, M., Universal sample preparation method for proteome analysis. Nature methods 2009, 6 (5), 359-62.