Sunday, July 24, 2011

Chlorambucil and nilotinib for end stage CLL?

A significant problem in treating CLL is that although patients often initially respond to conventional treatment they eventually become resistant to the drugs. While fludarabine based chemotherapy has become a commonly used therapy in CLL, chlorambucil (CLB) results in equivalent survival in older patients.

While we all know about TP53 deletions and mutations as causes of drug resistance in CLL, there are other mechanisms that are important.

CLB cytotoxicity is mediated by the introduction of DNA interstrand cross-links (ICLs). Interstrand crosslinking agents may induce double strand breaks (DSBs) as an intermediate step during ICL repair. Repair of CLB-induced DNA damage in CLL lymphocytes has been shown to be mediated by either nonhomologous end-joining (NHEJ) or homologous recombinational repair (HRR).

In other words alkylating agents like CLB damage cells by forming irregular links between the strands of DNA and as an intermediate step can cause simple breaks in the DNA strands. There are at least two mechanisms by which this DNA damage can be repaired.

Resistance to CLB treatment correlates with Rad51-related HRR or DNA-dependent protein kinase (DNA-PK) activity (a major component of NHEJ) in CLL lymphocytes. The HRR process is modulated by c-abl-mediated phosphorylation of Rad51. c-abl is important for normal B-cell development and is overexpressed in CLL-lymphocytes compared with normal B cells; the isoform 1b is involved in protection from apoptosis. CLB stimulates c-abl phosphorylation and activated c-abl results in phosphorylation of Rad51. Imatinib inhibition of c-abl results in decreased CLB-mediated c-abl phosphorylation and decreased CLB-mediated Rad51 phosphorylation with resultant sensitization of CLL lymphocytes to CLB.

As we have seen in the signaling series c-abl is an important molecule in CLL and I have previously written about imatinib (Gleevec) in CLL. In this month's Leukemia Research a paper from Canada looks again at CLB resistance and c-abl.

In this study they have demonstrated that the potent c-abl inhibitor nilotinib, when compared to imatinib, resulted in a greater synergistic effect with CLB in most of the CLL lymphocyte samples. Also, no statistically significant associations or differences were found between nilotinib or imatinib-mediated sensitization to CLB and the clinical status, Rai stage, Binet stage, ZAP70 expression, somatic mutations in IgVH or c-abl expression.

c-abl has the potential to become auto-phosphorylated on tyrosine and this correlates with increased activity. Biochemical assays with purified recombinant abl kinase showed that nilotinib is a 20–30-fold more potent inhibitor than imatinib for c-abl. Previously they showed that c-abl inhibition by imatinib resulted in sensitization to CLB in vitro. In support of these results, a phase I trial of imatinib and chlorambucil resulted in a 45% response rate in a heavily pretreated CLL patient population. Based on these results, they hypothesized that nilotinib in combination with CLB may be useful to improve therapy for CLL. In this in vitro study, nilotinib inhibited c-abl and sensitized primary B-CLL lymphocytes to CLB more than imatinib. The cytotoxicities of various drug combinations, obtained by MTT assay, were confirmed by analysis of the apoptotic pathway. Apoptosis induced by imatinib in cell lines of lymphatic origin is based on the cleavage of caspase-3. In the current study, They demonstrated that nilotinib potentiated CLB-induced caspase-3 activation to promote apoptotic cell death in primary CLL lymphocytes.

They have previously demonstrated that inhibition of DNA repair sensitizes CLL-lymphocytes to CLB. Down-regulation of either HRR or NHEJ pathways using specific inhibitors of c-abl (imatinib) or DNA-PK (NU7026/NU7441) respectively, resulted in an increase in CLB-induced cytotoxicity associated with decreased DNA repair. Evaluation of the HRR pathway confirmed their previous data showing that imatinib inhibited CLB-induced Rad51 foci formation. Surprisingly, in spite of the fact that nilotinib inhibits c-abl with higher potency than imatinib in CLL lymphocytes they did not find any differences between nilotinib and imatinib inhibition of CLB-induced Rad51 foci formation.

While one factor may be the sensitivity of the assay, their results strongly suggest that inhibition of c-abl with resulting inhibition of HRR can stimulate NHEJ. To evaluate if HRR inhibition can be compensated by the upregulation of the NHEJ pathway, they assessed the phosphorylation status of DNA-PK in CLL lymphocytes. In CLL lymphocyte samples where both c-abl inhibitors demonstrated similar sensitization to CLB, there was an increase in DNA-PK auto-phosphorylation(reflecting the activity of the kinase) induced by inhibition of CLB-induced HRR repair.

Furthermore, simultaneous inhibition of both the HRR (with a c-abl inhibitor; i.e. nilotinib or imatinib) and the NHEJ (with the DNA-PK inhibitor, NU7026) pathways potentiated the synergistic effect of either inhibitor alone on CLB cytotoxicity in CLL lymphocytes and was associated with an increase in CLB-induced DNA damage at 24 h and decreased DNA repair. The effect on CLB cytotoxicity in the presence of DNA-PK inhibition was more pronounced with the more potent c-abl inhibitor, nilotinib.

In conclusion, they have demonstrated a dynamic interaction between two major DNA repair pathways, HRR and NHEJ in CLL lymphocytes in response to CLB-induced DNA damage which should have important clinical implications.

The bottom line is that the the combination of nilotinib plus chlorambucil might be even better than Gleevec and chlorambucil for end-stage, drug resustant CLL. It wil be realtively expensive, but it does provide a possible last resort (oral) treatment. Clinical trials should begin shortly.

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