Deep sequencing reveals the spectrum of BCR-ABL1 mutations upon front-line therapy resistance in chronic myeloid leukemia: An Eastern-Indian cohort study.

The course of clinical management in chronic myeloid leukemia (CML) often faces a road-block in the form of front-line (imatinib) therapy resistance. Subsequently, several hotspot mutations were clinically validated in the kinase domain (KD) of BCR-ABL1, in deterring imatinib sensitivity and further, made targeted by next-generation tyrosine-kinase-inhibitor (TKI) drugs. Identifying KD mutations, occurring even at low frequencies, became pertinent here. Globally, cohorts from different origins were tested and the mutational spectra were mapped to categorize clinical management as well as related pathological features of CML. Moreover, targeted deep sequencing could reveal the mutational landscape more efficiently than the less sensitive Sanger sequencing method. However, no such efforts were reported from Eastern Indian cohorts of imatinib-resistant CML-sufferers. This study assessed a prospective study cohort of imatinib-resistant CML cases from Eastern India. Following dissecting the molecular and clinical parameters, the mutational spectrum was comparatively examined using conventional Sanger and next-generation deep sequencing method. This cohort showed a prevalence of e14a2-p210 variant of BCR-ABL1 and acquired resistance against imatinib, while the disease was mostly confined in its chronic phase. Together with a few common hotspot mutations identified in this cohort, deep sequencing revealed cases with a candidate mutation, otherwise undetermined by Sanger method. Also, cases with a second low frequency mutation were identified upon applying deep sequencing. Along with highlighting a few aspects of CML biology employing an Eastern-Indian cohort, this data could mark the immense importance of deep sequencing to contribute in the clinical management of CML upon front-line therapy resistance.

Long-Term Outcome of Philadelphia Chromosome Positive Leukemia From Eastern Indian Subcontinent: An Experience in the Era of Tyrosine Kinase Inhibitor (TKI) Therapy.

INTRODUCTION: Philadelphia chromosome (Ph) marks a group of leukemia with almost all cases of chronic myeloid leukemia (CML), a subset of acute lymphoid leukemia (ALL) and rare cases of acute myeloid leukemia (AML). In the era of precision medicine, such cases are successfully managed with tyrosine kinase inhibitor (TKI) drugs. This study examined the features and long-term outcome of Ph+ve cases from a tertiary cancer care center from Eastern Indian subcontinent. MATERIALS AND METHODS: Reviewing retrospective case-records registered between 2005 and 2015, cases of CML and ALL were documented under Ph+ve category; while no instance of Ph+ve AML was found. RESULTS: In CML cohort, adult and juvenile incidences were 95.2% and 4.8% respectively; in ALL cohort, the same was found for 66.67% and 33.33% cases. Among the CML cases, 10-year overall survival (OS) and progression-free survival (PFS) were significantly affected upon the phase of disease at time of detection. Furthermore, both OS and PFS significantly dropped whenever non-TKI-based treatment was applied prior to TKI-commencement. Long-term (10-year) sensitivity to 1st generation TKI, imatinib, was noted 88.51% and 83.33% for adult and juvenile CML cohorts, respectively. For Ph+ ALL cohort, the OS was benefitted upon combinatorial TKI and chemotherapy. However, large fractions of affected individual from CML as well as ALL cohorts were found to discontinue follow-up. CONCLUSION: Together with differences in outcome on the basis of drug-use from onset, age (juvenile versus adult) and stage at diagnosis, our analyses bring forward the real-world scenario of Ph+ve leukemia managed with precision medicine.

Visceral leishmaniasis masquerading as drug-induced pancytopenia in myasthenia gravis.

Visceral leishmaniasis (VL), also known as kala-azar (black fever in Hindi), is a disease primarily caused by Leishmania donovani. The most important clinical manifestation of visceral leishmaniasis is fever. Nonspecific laboratory findings of visceral leishmaniasis include anemia, neutropenia, eosinopenia, and thrombocytopenia. Definitive diagnosis of visceral leishmaniasis requires the demonstration of either parasite by smear or tissue by culture (usually bone marrow or spleen). Myasthenia gravis is an autoimmune disease caused by antibodies to acetylcholine receptors in the post-junctional membrane of the neuromuscular junction. It typically presents with fatigable muscle weakness without any sensory or brain involvement. It is usually treated with corticosteroids and immunosuppressants like azathioprine. Here we encountered a confirmed case of myasthenia gravis on azathioprine with pancytopenia. While working up to evaluate pancytopenia, bone marrow examination revealed presence of Donovan bodies and the patient showed good response to liposomal amphotericin-B. In retrospect, a case of myasthenia gravis, who presented with pancytopenia presumably drug-induced, was found to have visceral leishmaniasis.

Engineered domain swapping indicates context dependent functional role of RNA G-quadruplexes.

RNA domain swapping typically demonstrates conservation of the native function of the domain in a non-native context. In contrast, we employed RNA engineering to demonstrate deviation of G-quadruplex (GQ) function that is contingent upon its context dependent location, which is opposite to their native functional role. Known translation repressing RNA GQs were engineered into human VEGF IRES A replacing the endogenous GQ domain essential for translation. Alternatively, the translation inhibitory GQ motif within the 5'-UTR of MT3-MMP mRNA was replaced with two known GQ motifs that are essential for translation. The results indicate that the engineered GQ domains can adopt GQ structures in a foreign environment with a functional role reversal to accommodate the need of the endogenous swapped motifs. The observations establish the functionality and context dependent modularity of RNA GQ structures.

Metal Cations in G-Quadruplex Folding and Stability.

This review is focused on the structural and physicochemical aspects of metal cation coordination to G-Quadruplexes (GQ) and their effects on GQ stability and conformation. G-quadruplex structures are non-canonical secondary structures formed by both DNA and RNA. G-quadruplexes regulate a wide range of important biochemical processes. Besides the sequence requirements, the coordination of monovalent cations in the GQ is essential for its formation and determines the stability and polymorphism of GQ structures. The nature, location, and dynamics of the cation coordination and their impact on the overall GQ stability are dependent on several factors such as the ionic radii, hydration energy, and the bonding strength to the O6 of guanines. The intracellular monovalent cation concentration and the localized ion concentrations determine the formation of GQs and can potentially dictate their regulatory roles. A wide range of biochemical and biophysical studies on an array of GQ enabling sequences have generated at a minimum the knowledge base that allows us to often predict the stability of GQs in the presence of the physiologically relevant metal ions, however, prediction of conformation of such GQs is still out of the realm.

An Independently folding RNA G-quadruplex domain directly recruits the 40S ribosomal subunit.

In this study, we report that a 17-nucleotide independently folding RNA G-quadruplex (GQ) domain within the 294-nucleotide human VEGF IRES A interacts with the 40S ribosomal subunit. Footprinting and structure mapping analyses indicate that the RNA GQ forms independently and interacts directly with the 40S ribosomal subunit in the absence of other protein factors. Moreover, a filter binding assay in conjunction with enzymatic footprinting clearly established that the GQ-forming domain singularly dictates the binding affinity and also the function of internal ribosomal entry site (IRES) A. The deletion of the GQ domain abrogates the binding of the 40S ribosomal subunit to the IRES, which impairs cap-independent translation initiation. The findings provide a unique and defined role for a noncanonical RNA structure in cap-independent translation initiation by cellular IRESs. The GQ structure when present in an IRES acts as an essential element in contrast to their generally accepted inhibitory role in translation. The results of this study explain the hitherto unknown mechanistic necessity of the GQ structure in IRES function.

Rationally induced RNA:DNA G-quadruplex structures elicit an anticancer effect by inhibiting endogenous eIF-4E expression.

RNA G-quadruplex (GQ) structures act as regulators of a diverse array of cellular processes including translation, pre-mRNA processing, and mRNA targeting. We report here a strategy of harnessing the natural ability of RNA GQs to inhibit translation by rationally inducing a GQ on a targeted mRNA to knockdown endogenous gene expression. We chose to target eIF-4E because of its key role in translation initiation and overexpression in multiple cancers and with the expectation that downregulation of eIF-4E would result in antiproliferation of cancer cells. Targeted hybrid (RNA:DNA) GQ structures were induced at the 5'-untranslated region (UTR) and the protein coding region of the eIF-4E mRNA by rationally designed and partially modified extraneous DNA sequences and their effect on eIF-4E expression was determined. The formation of a stable induced G-quadruplex was established by biophysical and biochemical methods. Thermodynamic parameters calculated from CD melting indicate formation of a stable induced GQ at a physiologically relevant salt concentration. We established the specificity and efficacy of the induced GQ formation by monitoring the targeted repression of a reporter gene. Most importantly we have demonstrated that inducing GQ in the 5'-UTR and the protein coding region of eIF-4E mRNA in human cancer cells results in 30% and 60% inhibition of the endogenous protein expression, respectively. Treating with the GQ inducing oligonucleotide sequences resulted in a decrease in the viability of human cancer cells in a dose-dependent manner. The above concept opens up a new strategy for targeted modulation of endogenous gene expression.