Arunima Mukhopadhyay

ATG7 regulates energy metabolism, differentiation and survival of Philadelphia-chromosome-positive cells

ABSTRACT A major drawback of tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) is that primitive CML cells are able to survive TKI-mediated BCR-ABL inhibition, leading to disease persistence in patients. Investigation of strategies aiming to inhibit alternative survival pathways in CML is therefore critical. We have previously shown that a nonspecific pharmacological inhibition of autophagy potentiates TKI-induced death in Philadelphia chromosome-positive cells. Here we provide further understanding of how specific and pharmacological autophagy inhibition affects nonmitochondrial and mitochondrial energy metabolism and reactive oxygen species (ROS)-mediated differentiation of CML cells and highlight ATG7 (a critical component of the LC3 conjugation system) as a potential specific therapeutic target. By combining extra- and intracellular steady state metabolite measurements by liquid chromatography-mass spectrometry with metabolic flux assays using labeled glucose and functional assays, we demonstrate that knockdown of ATG7 results in decreased glycolysis and increased flux of labeled carbons through the mitochondrial tricarboxylic acid cycle. This leads to increased oxidative phosphorylation and mitochondrial ROS accumulation. Furthermore, following ROS accumulation, CML cells, including primary CML CD34+ progenitor cells, differentiate toward the erythroid lineage. Finally, ATG7 knockdown sensitizes CML progenitor cells to TKI-induced death, without affecting survival of normal cells, suggesting that specific inhibitors of ATG7 in combination with TKI would provide a novel therapeutic approach for CML patients exhibiting persistent disease.

Autophagy in Chronic Myeloid Leukaemia: Stem Cell Survival and Implication in Therapy

The insensitivity of Chronic Myeloid Leukaemia (CML) stem cells to Tyrosine Kinase Inhibitor (TKI) treatment is now believed to be the main reason for disease persistence experienced in patients. It has been shown that autophagy, an evolutionarily conserved catabolic process that involves degradation of unnecessary or harmful cellular components via lysosomes, is induced following TKI treatment in CML cells. Of clinical importance, autophagy inhibition, using the anti-malarial drug hydroxychloroquine (HCQ), sensitised CML cells, including primitive CML stem cells, to TKI treatment. In this review we discuss the role of autophagy in the maintenance and survival of stem cells in more detail, with a focus on its role in survival of CML stem cells and the possibility to inhibit this pathway as a way to eliminate persistent CML stem cells in vitro and in patients.

Hydroxychloroquine for chronic myeloid leukemia: complete cure on the horizon?

Chronic myeloid leukemia (CML) is characterized by a reciprocal translocation (t[9;22][q34;q11]) between chromosomes 9 and 22, producing the Philadelphia chromosome. This abnormal chromosome carries a fusion gene, BCR–ABL, that is translated into the constitutively active BCR–ABL protein tyrosine kinase (TK). In hemopoietic stem cells, this protein activates a cascade of signaling pathways resulting in increased survival, proliferation [1], altered adhesion [2] and limited growth factor dependence. The progeny of these cells outgrow the normal cells to propagate the disease, CML. Functionally, protein kinases interact with ATP and the introduction of an ATP competitive inhibitor of BCR–ABL kinase was a major therapeutic milestone for CML. In 1998, the first TK inhibitor (TKI), imatinib, was introduced to the clinic. Consequently, ABL kinase-targeted [3] small-molecule inhibitors captured the market, with the consecutive development of broad-spectrum inhibitors such as dasatinib and more potent inhibitors such as nilotinib.

The Toxoplasma gondii plastid replication and repair enzyme complex, PREX

A plastid-like organelle, the apicoplast, is essential to the majority of medically and veterinary important apicomplexan protozoa including Toxoplasma gondii and Plasmodium. The apicoplast contains multiple copies of a 35 kb genome, the replication of which is dependent upon nuclear-encoded proteins that are imported into the organelle. In P. falciparum an unusual multi-functional gene, pfprex, was previously identified and inferred to encode a protein with DNA primase, DNA helicase and DNA polymerase activities. Herein, we report the presence of a prex orthologue in T. gondii. The protein is predicted to have a bi-partite apicoplast targeting sequence similar to that demonstrated on the PfPREX polypeptide, capable of delivering marker proteins to the apicoplast. Unlike the P. falciparum gene that is devoid of introns, the T. gondii prex gene carries 19 introns, which are spliced to produce a contiguous mRNA. Bacterial expression of the polymerase domain reveals the protein to be active. Consistent with the reported absence of a plastid in Cryptosporidium species, in silico analysis of their genomes failed to demonstrate an orthologue of prex. These studies indicate that prex is conserved across the plastid-bearing apicomplexans and may play an important role in the replication of the plastid genome.

Targeting BCR-ABL-Independent TKI resistance in chronic myeloid leukemia by mTOR and autophagy inhibition

Abstract Background Imatinib and second-generation tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib have statistically significantly improved the life expectancy of chronic myeloid leukemia (CML) patients; however, resistance to TKIs remains a major clinical challenge. Although ponatinib, a third-generation TKI, improves outcomes for patients with BCR-ABL-dependent mechanisms of resistance, including the T315I mutation, a proportion of patients may have or develop BCR-ABL-independent resistance and fail ponatinib treatment. By modeling ponatinib resistance and testing samples from these CML patients, it is hoped that an alternative drug target can be identified and inhibited with a novel compound. Methods Two CML cell lines with acquired BCR-ABL-independent resistance were generated following culture in ponatinib. RNA sequencing and gene ontology (GO) enrichment were used to detect aberrant transcriptional response in ponatinib-resistant cells. A validated oncogene drug library was used to identify US Food and Drug Administration–approved drugs with activity against TKI-resistant cells. Validation was performed using bone marrow (BM)–derived cells from TKI-resistant patients (n = 4) and a human xenograft mouse model (n = 4–6 mice per group). All statistical tests were two-sided. Results We show that ponatinib-resistant CML cells can acquire BCR-ABL-independent resistance mediated through alternative activation of mTOR. Following transcriptomic analysis and drug screening, we highlight mTOR inhibition as an alternative therapeutic approach in TKI-resistant CML cells. Additionally, we show that catalytic mTOR inhibitors induce autophagy and demonstrate that genetic or pharmacological inhibition of autophagy sensitizes ponatinib-resistant CML cells to death induced by mTOR inhibition in vitro (% number of colonies of control[SD], NVP-BEZ235 vs NVP-BEZ235+HCQ: 45.0[17.9]% vs 24.0[8.4]%, P = .002) and in vivo (median survival of NVP-BEZ235- vs NVP-BEZ235+HCQ-treated mice: 38.5 days vs 47.0 days, P = .04). Conclusion Combined mTOR and autophagy inhibition may provide an attractive approach to target BCR-ABL-independent mechanism of resistance.

Targeting quiescent leukemic stem cells using second generation autophagy inhibitors

In chronic myeloid leukemia (CML), tyrosine kinase inhibitor (TKI) treatment induces autophagy that promotes survival and TKI-resistance in leukemic stem cells (LSCs). In clinical studies hydroxychloroquine (HCQ), the only clinically approved autophagy inhibitor, does not consistently inhibit autophagy in cancer patients, so more potent autophagy inhibitors are needed. We generated a murine model of CML in which autophagic flux can be measured in bone marrow-located LSCs. In parallel, we use cell division tracing, phenotyping of primary CML cells, and a robust xenotransplantation model of human CML, to investigate the effect of Lys05, a highly potent lysosomotropic agent, and PIK-III, a selective inhibitor of VPS34, on the survival and function of LSCs. We demonstrate that long-term haematopoietic stem cells (LT-HSCs: Lin−Sca-1+c-kit+CD48−CD150+) isolated from leukemic mice have higher basal autophagy levels compared with non-leukemic LT-HSCs and more mature leukemic cells. Additionally, we present that while HCQ is ineffective, Lys05-mediated autophagy inhibition reduces LSCs quiescence and drives myeloid cell expansion. Furthermore, Lys05 and PIK-III reduced the number of primary CML LSCs and target xenografted LSCs when used in combination with TKI treatment, providing a strong rationale for clinical use of second generation autophagy inhibitors as a novel treatment for CML patients with LSC persistence.

Comparison of prophylactic and therapeutic immunisation with an ErbB-2 (HER2) fusion protein and immunoglobulin V-gene repertoire analysis in a transgenic mouse model of spontaneous breast cancer

ErbB-2 is associated with several solid tumours of which breast cancer is the commonest cancer in women worldwide. Though anti-ErbB-2 antibody appears to play a significant role in prevention and therapy, naturally occurring anti-ErbB-2 antibody associated with the cleaved ectodomain of overexpressed ErbB-2 self antigen is detectable in patients. It is therefore essential to understand the course of antibody mediated protection during disease progression. 100% of FVB/N(neu) mice expressing mutated, constitutively active ErbB-2 develop mammary carcinoma. It has been shown that vaccination with ErbB-2 associated with a T helper cell epitope P30 can offer protection against transplantable tumour but it is unclear whether the same vaccine protects against naturally developing tumour. We have analysed the course of the disease following prophylactic, and therapeutic vaccination in this spontaneous, eutopic mammary carcinoma model that more closely resembles the human disease. 100% protection against tumour development was observed subsequent to prophylactic immunisation but disease progression was unaffected by therapeutic vaccination. The antibody response exhibited restricted expansion of the Immunoglobulin (Ig) variable (V)-gene repertoire by ErbB-2 specific B cells compared with the non-antigen specific B cell pool and control mice. The serum antibody profile was similar in therapeutically injected mice without any effect on tumour burden.