Leprosy drug Clofazimine activates peroxisome proliferator-activated receptor-γ and synergizes with Imatinib to inhibit chronic myeloid leukemia cells

Leukemia stem cells contribute to drug-resistance and relapse in chronic myeloid leukemia and BCR-ABL1 inhibitor monotherapy fails to eliminate them, thereby necessitating alternate therapeutic strategies. Peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone downregulates signal transducer and activator of transcription 5 (STAT5) and in combination with imatinib induces complete molecular response in imatinib-refractory patients by eroding leukemia stem cells. Thiazolidinediones like pioglitazone however, are associated with severe side effects. To identify alternate therapeutic strategies for chronic myeloid leukemia, screened FDA-approved drugs in K562 cells and identified the leprosy drug clofazimine as an inhibitor of viability. It is showed that clofazimine induces apoptosis in chronic myeloid leukemia patient-derived blood mononuclear cells, with particularly robust effect in imatinib-resistant cells. Clofazimine also induced apoptosis in CD34+38- progenitors and quiescent CD34+ cells from chronic myeloid leukemia patients but not healthy donor-derived hematopoietic progenitors. Mechanistic evaluation revealed that clofazimine via physical interaction with PPARγ induced nuclear factor kB-p65 proteasomal degradation, which led to sequential MYB and peroxiredoxin 1 downregulation and concomitant induction of reactive oxygen species-mediated apoptosis. Clofazimine also suppressed STAT5 expression and consequently downregulated stem cell maintenance factors hypoxia-inducible factor -1α and -2α and Cbp/P300 Interacting Transactivator with Glu/Asp rich Carboxy-Terminal Domain 2. Combining imatinib with clofazimine caused a far superior synergy than pioglitazone where clofazimine reduced imatinib's IC50 by >4 logs and remarkably eroded quiescent CD34+ cells. In a K562 xenograft study clofazimine and imatinib co-treatment showed more robust efficacy than individual treatments. We propose clinical evaluation of clofazimine in imatinib-refractory chronic myeloid leukemia. (Haematologica 2019; pii: haematol.2018.194910. doi: 10.3324/haematol.2018.194910).

CDK2 destabilizes tumor suppressor C/EBPα expression through ubiquitin-mediated proteasome degradation in acute myeloid leukemia

Deregulation and functional inhibition of C/EBPα, a key transcription factor of myeloid lineage leads to development of myeloid leukemia. It was shown that CDK2 negatively regulates C/EBPα protein levels in myeloid leukemia cells. Overexpression of CDK2 inhibited C/EBPα both in a heterologous HEK293T and U937 myeloid leukemia cells. On the contrary, CDK2 depletion enhanced endogenous C/EBPα protein levels. CDK2 mitigated C/EBPα levels by promoting its ubiquitin-mediated proteasome degradation. It is further showed that although CDK2 interacted with C/EBPα, direct interaction of CDK2 with C/EBPα is not involved in C/EBPα downregulation. CDK2-dependent phosphorylation of C/EBPα on its widely reported phosphorylatable amino acid residues is apparently not required for C/EBPα degradation by CDK2. Furthermore, This data demonstrate that CDK2-driven C/EBPα inhibition mitigates its transactivation potential and cellular functions such as ability to promote myeloid differentiation and growth arrest (J Cell Biochem. 2019; doi: 10.1002/jcb.29516).

Direct physical interaction of active Ras with mSIN1 regulates mTORC2 signaling

The mechanistic (or mammalian) target of rapamycin (mTOR), a Ser/Thr kinase, associates with different subunits forming two functionally distinct complexes, mTORC1 and mTORC2, regulating a diverse set of cellular functions in response to growth factors, cellular energy levels, and nutrients. The mechanisms regulating mTORC1 activity are well characterized; regulation of mTORC2 activity, however, remains obscure. While studies conducted in Dictyostelium suggest a possible role of Ras protein as a potential upstream regulator of mTORC2, definitive studies delineating the underlying molecular mechanisms, particularly in mammalian cells, are still lacking. Protein levels were measured by Western blotting and kinase activity of mTORC2 was analyzed by in vitro kinase assay. In situ Proximity ligation assay (PLA) and co-immunoprecipitation assay was performed to detect protein-protein interaction. Protein localization was investigated by immunofluorescence and subcellular fractionation while cellular function of mTORC2 was assessed by assaying extent of cell migration and invasion. Earlier, presented experimental evidence in support of the role of Ras activation as an upstream regulatory switch governing mTORC2 signaling in mammalian cancer cells. Herein, report that active Ras through its interaction with mSIN1 accounts for mTORC2 activation, while disruption of this interaction by genetic means or via peptide-based competitive hindrance, impedes mTORC2 signaling. This study defines the regulatory role played by Ras during mTORC2 signaling in mammalian cells and highlights the importance of Ras-mSIN1 interaction in the assembly of functionally intact mTORC2 .(BMC Cancer. 2019; 19(1):1236)

7-hydroxyfrullanolide, isolated from Sphaeranthus indicus, inhibits colorectal cancer cell growth by p53-dependent and -independent mechanism

Sphaeranthus indicus Linn. is commonly used in Indian traditional medicine for management of multiple pathological conditions. However, there are limited studies on anticancer activity of this plant and its underlying molecular mechanisms. An active constituent, 7-hydroxyfrullanolide (7-HF) was isolated from the flowers of this plant, which showed promising chemotherapeutic potential. The compound was more effective in inhibiting in vitro proliferation of colon cancers cells through G2/M phase arrest than other cancer cell lines that were used in this study. Consistent with in vitro data, 7-HF caused substantial regression of tumour volume in a syngeneic mouse model of colon cancer. The molecule triggered extrinsic apoptotic pathway, which was evident as upregulation of DR4 and DR5 expression as well as induction of their downstream effector molecules (FADD, Caspase-8). Concurrent activation of intrinsic pathway was demonstrated with loss of ΔΨm to release pro-apoptotic cytochrome c from mitochondria and activation of downstream caspase cascades (Caspase -9, -3). Loss of p53 resulted in decreased sensitivity of cells towards pro-apoptotic effect of 7-HF with increased number of viable cells indicating p53-dependent arrest of cancer cell growth. This notion was further supported with 7-HF-mediated elevation of endogenous p53 level, decreased expression of MDM2 and transcriptional upregulation of p53 target genes in apoptotic pathway. However, 7-HF was equally effective in preventing progression of HCT116 p53+/+ and p53-/- cell derived xenografts in nude mice, which suggests that differences in p53 status may not influence its in vivo efficacy. Taken together, these results support 7-HF as a potential chemotherapeutic agent and provided a new mechanistic insight into its anticancer activity (Carcinogenesis. 2019; 40(6): 791-804).

Microtubule disrupting agent‐mediated inhibition of cancer cell growth is associated with blockade of autophagic flux and simultaneous induction of apoptosis

Given that autophagy inhibition is a feasible way to enhance sensitivity of cancer cells towards chemotherapeutic agents, identifying potent autophagy inhibitor has obvious clinical relevance. In this study, investigated ability of TN‐16, a microtubule disrupting agent, on modulation of autophagic flux and its significance in promoting in vitro and in vivo cancer cell death. The effect of TN‐16 on cancer cell proliferation, cell division, autophagic process and apoptotic signalling was assessed by various biochemical (Western blot and SRB assay), morphological (TEM, SEM, confocal microscopy) and flow-cytometric assays. In vivo anti‐tumour efficacy of TN‐16 was investigated in syngeneic mouse model of breast cancer. TN‐16 inhibited cancer cell proliferation by impairing late‐stage autophagy and induction of apoptosis. Inhibition of autophagic flux was demonstrated by accumulation of autophagy‐specific substrate p62 and lack of additional LC3‐II turnover in the presence of lysosomotropic agent. The effect was validated by confocal micrographs showing diminished autophagosome‐lysosome fusion. Further studies revealed that TN‐16–mediated inhibition of autophagic flux promotes apoptotic cell death. Consistent with in vitro data, results of this in vivo study revealed that TN‐16–mediated tumour growth suppression is associated with blockade of autophagic flux and enhanced apoptosis. This data signify that TN‐16 is a potent autophagy flux inhibitor and might be suitable for (pre‐) clinical use as standard inhibitor of autophagy with anti‐cancer activity (Cell Proliferation. 2019; in Press)

Androgen deprivation upregulates SPINK1 expression and potentiates cellular plasticity in prostate cancer

Emergence of aggressive neuroendocrine prostate cancer (NEPC) associated with androgen-deprivation therapy (ADT) has been known. Despite its adverse clinical effects, majority of advanced-stage prostate cancer (PCa) patients including SPINK1-positive subtype are subjected to ADT. It was shown that androgen receptor (AR) and its corepressor, REST, functions as transcriptional-repressor of SPINK1, and AR-antagonists alleviate this repression leading to SPINK1 upregulation. Moreover, increased SOX2 levels during NE-trans-differentiation transactivates SPINK1, a critical player in maintenance of NE-phenotype. Additionally, SPINK1 elicits epithelial-mesenchymal-transition, stemness, drug-resistance and cellular-plasticity. Conversely, pharmacological Casein Kinase-1 inhibition stabilizes the REST levels, which in cooperation with AR conjures SPINK1 transcriptional-repression and impedes SPINK1-mediated oncogenesis. Notably, elevated levels of SPINK1 and NEPC markers were observed in tumors of AR-antagonists treated mice xenograft models, and in a subset of NEPC patients. Collectively, these findings provide a plausible explanation to the paradoxical clinical-outcomes of ADT, possibly due to SPINK1 upregulation, and offers strategy for adjuvant-therapies (Nat. Comm. 2020; 11(1); 384)