Babu Rao Vundinti

Recent developments in drug resistance mechanism in chronic myeloid leukemia: a review

A revolution in medical science was marked with the advent of imatinib, a site‐specific drug for the management of patients with chronic myeloid leukemia (CML). Imatinib mesylate (also known as Glivec, Gleevec, STI‐571, CGP57148), an orally administered 2‐phenylaminopyrimidine derivative approved by FDA in 2001 for the treatment for CML, is highly effective in treating the early stages of CML, but remission induced in advanced phase was observed to be relatively short‐lived. The primary cause of resistance in patients with CML is the mutations in the BCR–ABL kinase domain. This review discusses the different mechanisms leading to imatinib resistance and various treatment options to over‐ride imatinib resistance.

Down-regulation of miR-199b associated with imatinib drug resistance in 9q34.1 deleted BCR/ABL positive CML patients.

Chronic myeloid leukemia (CML) occurs due to t(9,22) (q34;q11) and molecularly BCR/ABL gene fusion. About 15-18% Philadelphia positive CML patients have gene deletions around the translocation breakpoints on 9q34.1. The microRNAs (miRNAs), namely miR-219-2 and miR-199b, centromeric to the ABL1 gene are frequently lost in CML patients. We have designed a study to determine miR-219-2 and miR-199b expression levels which would help to understand the prognosis of imatinib therapy. A total of 150 CML patients were analyzed to identify 9q deletion. Fluorescent in-situ hybridization (FISH) was performed using BCR/ABL dual color, dual fusion probe to study the signal pattern and BAC probes for miR-199b and miR-219-2 (RP11-339B21 and RP11-395P17) to study the miRNA deletions. The expression level of miRNA was analyzed by real-time polymerase chain reaction (RT-PCR). FISH analysis revealed 9q34.1 deletion in 34 (23%) CML patients. The deletions were not detected using BAC probes for miRNAs in 9q deleted patients. The expression analysis showed down-regulation of miR-199b and miR-219-2 in the 9q deleted patients (34 CML) as compared to a pool of patients without deletion. However, miR-199b (9q34.11) was significantly (p=0.001) down-regulated compared to miR-219-2. The follow-up study showed that the miR-199b was found to be strongly associated with imatinib resistance, as 44.11% patients showed resistance to imatinib therapy. Hence, the deletion in 9q34.1 region (ABL) plays an important role in disease pathogenesis. Eventually, miRNAs can provide new therapeutic strategies and can be used as a prognostic indicator.

Cytogenetic abnormalities in myelodysplastic syndrome: an overview

Abstract The myelodysplastic syndromes (MDS) are clonal disorders of haematopoietic stem cells characterized by ineffective haematopoiesis leading to blood cytopenias and by high incidence of progression to acute myeloid leukaemia (AML). These disorders generally arise de novo, but may also occur years after exposure to mutagenic chemotherapy. Clonal cytogenetic abnormalities are detected in about 30–50% de novo cases, whereas more than 80% of therapy-related forms harbour such markers. Although in the Western countries, MDS cases are mainly reported in the elderly population and rarely in the paediatric age group; this disease is increasingly seen in young adults in India. Cytogenetic study plays an important role in the diagnosis and is useful for prediction of individual prognosis using the international prognostic scoring system. Specific chromosomal abnormalities, such as −5/5q-, −7/7q-, and complex abnormalities, play an important role in the development of new therapeutic options and clinical management of MDS. In this review, we summarize the cytogenetic abnormalities in MDS from various parts of the world.

Current and emerging therapeutic strategies for Fanconi anemia

Fanconi Anemia (FA) is a rare disorder with incidence of 1in 350,000 births. It is characterized by progressive bone marrow failure leading to death of many patients in their childhood while development of cancer at later stages of life in some. The treatment of FA is still a medical challenge. Current treatments of FA include androgen administration, hematopoietic growth factors administration and hematopoietic stem cell transplantation (HSCT). Clinical gene therapy trials are still ongoing. The partial success of current therapies has renewed interest in the search for new treatments. Generation of patient-specific induced pluripotent stem (iPS) has shown promising results for cell and gene based therapy. Small molecule interventions have been observed to delay tumor onset in FA. Tumors deficient in FA pathway can be treated by profiling of DNA repair pathway through synthetic lethality mechanism. Targeting toll-like receptor 8 (TLR8) dependent TNFα overexpression is yet another upcoming therapeutic approach to treat FA patients. In conclusion, in the present scenario of treatments available for FA, a proper algorithm of treatment decisions must be followed for better management of FA patients and to ensure their increased survival. Innovative therapeutic approaches that can prevent both anemia and cancer should be developed for more effective treatment of FA.

Clinical, genetic and cytogenetic study of Fanconi anemia in an Indian population

Abstract Fanconi anemia (FA) is a rare autosomal recessive genetic disease, associated with congenital anomalies and a predisposition to cancers. FA patients exhibit spontaneous chromosome breakage and FA cells are sensitive to DNA interstrand crosslink agents and expresses high frequency of chromosome breakage. Recently 13 genes have been shown to be involved with the FA phenotype. We have carried out a detailed study in clinically diagnosed FA patients in an Indian population. Thirty three patients were clinically diagnosed with FA and had aplastic anemia and bleeding abnormalities. The genetic analysis revealed a significantly (P<0·0001) high frequency (36·4%) of parental consanguinity in FA patients compared to controls (3·33%). Chromosomal analysis revealed spontaneous chromosome breakage in 63·64% FA patients. The mitomycin C and diepoxybutane induced cultures showed a significantly (P<0·001) high frequency of chromosome breakage and radial formation compared to controls. Among 33 patients, nine (27·27%) patients developed malignancies and chromosomal abnormalities were detected in five (55·5%) patients bone marrow cells including monosomy 5 and 7, trisomy 10, der(1q) and inv(7). Cytogenetic investigation is important in aplastic anemia to rule out FA. The clinical presentation and the associated high frequency of consanguinity in FA, and the molecular analysis are complementary in the study of an Indian population.

Three way translocation in a new variant of t(8;21) acute myeloid leukemia involving Xp22.

The t(8;21)(q22;q22) is one of the most frequent chromosomal abnormality associated with acute myeloid leukemia (AML) M2 sub type. The additional chromosomal abnormalities including structural and numerical are frequently reported with the translocation, t (8;21)(q22;q22). We report a case of AML-M2 with t(X;8;21)(p22;q22;q22) associated with loss of Y chromosome. Using a dual color fluorescence in situ hybridization (FISH) analysis with ETO and AML1 probes, we demonstrated an ETO/AML1 fusion signal on the derivative chromosome 8 and one ETO signal on derivative Chromosome Xp22. The patient did not respond to therapy and follow-up of cytogenetics revealed same chromosome abnormality. Hence, this three way translocation involving X chromosome might be associated with poor prognosis.

Genetic variations of hOCT1 gene and CYP3A4/A5 genes and their association with imatinib response in Chronic Myeloid Leukemia

There is an increasing body of evidence demonstrating that mechanisms independent of BCR/ABL gene also contribute to imatinib resistance in Chronic Myeloid Leukemia (CML). It has been extensively reported that polymorphisms of the genes associated with imatinib metabolization and imatinib influx/efflux play an important role in the disease resistance. We investigated the impact of 12 genetic variants of the two genes, CYP3A4/A5 and the human cation transporter 1 gene (hOCT1) on the clinical outcome, in a cohort of 106 newly diagnosed CML patients. In the patient cohort investigated, only 6 variant alleles could be detected. The others were not present and could not be investigated. Two polymorphisms, CYP3A5*3 (rs776746)and hOCT1 M408V (rs628031), were significantly associated with the Complete Cytogenetic Response (CCyR) at 6 months and Major Molecular Response (MMR) at 12 months. The presence of favourable alleles at M408V and M420del in combination was associated with a MMR at 12 months. Functional polymorphisms of the genes associated with imatinib influx and metabolization may play a role in predicting primary response to imatinib and treatment outcome.

Evolution of BCR/ABL Gene Mutation in CML Is Time Dependent and Dependent on the Pressure Exerted by Tyrosine Kinase Inhibitor

Background Mutations in the ABL kinase domain and SH3-SH2 domain of the BCR/ABL gene and amplification of the Philadelphia chromosome are the two important BCR/ABL dependent mechanisms of imatinib resistance. Here, we intended to study the role played by TKI, imatinib, in selection of gene mutations and development of chromosomal abnormalities in Indian CML patients. Methods Direct sequencing methodology was employed to detect mutations and conventional cytogenetics was done to identify Philadelphia duplication. Results Among the different mechanisms of imatinib resistance, kinase domain mutations (39%) of the BCR/ABL gene were seen to be more prevalent, followed by mutations in the SH3-SH2 domain (4%) and then BCR/ABL amplification with the least frequency (1%). The median duration of occurrence of mutation was significantly shorter for patients with front line imatinib than those pre-treated with hydroxyurea. Patients with high Sokal score (p = 0.003) showed significantly higher incidence of mutations, as compared to patients with low/intermediate score. Impact of mutations on the clinical outcome in AP and BC was observed to be insignificant. Of the 94 imatinib resistant patients, only 1 patient exhibited duplication of Philadelphia chromosome, suggesting a less frequent occurrence of this abnormality in Indian CML patients. Conclusion Close monitoring at regular intervals and proper analysis of the disease resistance would facilitate early detection of resistance and thus aid in the selection of the most appropriate therapy.

DNA interstrand cross‐link repair: understanding role of Fanconi anemia pathway and therapeutic implications

Interstrand cross‐links (ICLs) are extremely toxic DNA lesions that prevent DNA double‐helix separation due to the irreversible covalent linkage binding of some agents on DNA strands. Agents that induce these ICLs are thus widely used as chemotherapeutic drugs but may also lead to tumor growth. Fanconi anemia (FA) is a rare genetic disorder that leads to ICL sensitivity. This review provides update on current understanding of the role of FA proteins in repairing ICLs at various stages of cell cycle. We also discuss link between DNA cross‐link genotoxicity caused by aldehydes in FA pathway. Besides this, we summarize various ICL agents that act as drugs to treat different types of tumors and highlight strategies for modulating ICL sensitivity for therapeutic interventions that may be helpful in controlling cancer and life‐threatening disease, FA.

A triad of telomerase, androgen receptor and early growth response 1 in prostate cancer cells

ABSTRACT Telomerase activation is one of the key mechanisms that allow cells to bypass replicative senescence. Telomerase activity is primarily regulated at the level of transcription of its catalytic unit- hTERT. Prostate cancer (PCa), akin to other cancers, is characterized by high telomerase activity. Existing data suggest that hTERT expression and telomerase activity are positively regulated by androgenic stimuli in androgen-dependent prostate cancer (ADPC) cells. A part of the present study reaffirmed this by demonstrating a decline in the hTERT expression and telomerase activity on “loss of AR” in ADPC cells. The study further addressed 2 unresolved queries, i) whether AR-mediated signaling is of any relevance to hTERT expression in castration-resistant prostate cancer (CRPC) and ii) whether this signaling involves EGR1. Our data suggest that AR-mediated signaling negatively regulates hTERT expression in CRPC cells. Incidental support for the possibility of EGR1 being a regulator of hTERT expression in PCa was provided by i) immunolocalization of hTERT and EGR1 proteins in the same cell type (secretory epithelium) of PCa and BPH tissues; ii) significantly (p< 0.001) higher levels of both these proteins in CRPC (PC3 and DU145), compared with ADPC (LNCaP) cells. A direct evidence for the role of EGR1 in hTERT expression was evident by a significant (p<0.0001) decrease in the hTERT transcript levels in the EGR1-silenced CRPC cells. Further, “gain of AR” led to a significant reduction in the levels of hTERT and EGR1 in CRPC cells. However, restoration of EGR1 levels prevented the decline in the hTERT transcript levels in these cells. Taken together, our data indicate that AR regulates the expression of EGR1, which in turn acts as a positive regulator of hTERT expression in CRPC cells. Thus, AR exerts an inhibitory effect on hTERT expression and telomerase activity by modulating EGR1 levels in CRPC cells.