(a) Identification of deregulated E3 ubiquitin ligases and Deubiquitinases (DUBs) and their impact on stability of key regulatory proteins (oncogenes, tumor suppressor and TFs) implicated in disease manifestations.

E3 ubiquitin ligases (E3s) regulate a variety of biologic processes by timely ubiquitination and degradation of many cell cycle and apoptosis- regulatory proteins. These E3 ubiquitin ligases transfer activated ubiquitin moieties in the ubiquitination process from Ubiquitin E2 ligases to the substrates that are destined to be degraded. Finally, these polyubiquitinated protein substrates are degraded by proteasomes where Deubiquitinases (DUBs) remove ubiquitin moieties (that becomes available as free pool for E3s) prior to their degradation by proteasomes. Since E3s timely regulate protein turnover, their controlled regulation is crucial for cell functioning. Due to their common deregulation and a role in the development of cancers, targeting the ubiquitin cascade (Fig. 1) in cancer therapeutics has emerged as a promising approach. In fact, FDA approval of general proteasome inhibitor bortezomib in the treatment of multiple myeloma has established ubiquitin-protasome system as a potential therapeutic target in cancer therapy. Our Lab focuses to understand mechanisms leading to deregulation of key E3s viz. FBW7, SKP2 and E6AP (at mRNA as well as protein levels), identify their substrates and elucidate how these pathways can be targeted for therapeutic benefit. Our research has identified several new substrates of FBW7 as well as E6AP not known earlier. Currently, we are engaged in identifying how FBW7 is regulated at promoter levels.

(b) Unravelling mechanisms underlying downregulation and functional inhibition of master myeloid transcription factors C/EBPα and PU.1 in Acute myeloid leukemia.

Hematopoiesis is the formation of mature functional blood cells from hematopoietic stem cells through a series of developmental process. Deregulation of this process often culminates in Leukemia which is characterized by accumulation abnormal blood cells. Leukemia arises due to sequential accumulation of mutations in stem or progenitor cells which initially gives rise to preleukemic condition and transforms to leukemia upon acquisition of additional mutations. Accumulating mutations provide stemness and self renewal to these progenitor cells which do not mature (i.e do not differentiate to functional cells) and keep on accumulating. Process of differentiation in these cells is governed by lineage specific transcription factors which play key role in regulating expression of factors involved in acquiring molecular and phenotypic changes along a particular lineage. C/EBPα and PU.1 are key transcription factors of myeloid lineage and their dysregulation is an established leukemogenic event. Although current AML treatment majorly involves intensive chemotherapy, such patients often relapse. On the other hand, APL (acute promyelocyticleukemia) and CML (chronic myeloid leukemia) are treated with targeted therapy where ATRA and Imatinib mesylate respectively have better therapeutic output. Owing to better therapeutic outcome, differentiation is preferred, however, Majority of AML (other than APL and CML) are non-responsive to differentiation therapy. Interestingly, in majority of AML C/EBPα and PU.1 are functionally inhibited. Elucidating mechanisms underlying inhibition of these factors could be therapeutically targeted to induce differentiation. We therefore utilize proteomic and transcriptomic approach to identify interacting protein partners of these transcription factors to understand their biological significance. Recently we identified E6AP as well as SKP2 E3 ligases that negatively regulate C/EBPα protein stability and its functions; in addition, we also showed Fbw7, a component of SCF ubiquitin ligase (SCFFbw7), in cooperation with GSK3β may negatively modulate G-CSFR protein steady state levels by promoting its degradation and thus critically regulates G-CSFR signaling. However, as there may be several E3s for a given substrate, we intend to identify other E3 ubiquitin ligases/Deubiquitinases that negatively regulate these key regulators (C/EBPα, GCSFr, PU.1 and AML1) leading to differentiation arrest in myeloid cells.

(c) Understanding FLT3/FLT3-ITD signaling in AML for therapeutic targeting:

FLT3 (fms-like tyrosine kinase 3) is a tyrosine receptor kinase expressed on the cell surface of myeloid progenitors, FLT3 ligand (FL) biding to this receptor activates it leading to proliferation and survival of these myeloid cells. However, in majority of AML (>35%) cases this receptor is mutated that makes it constitutively active. Internal tandem duplication (FLT3-ITD) mutations in juxtamembrane domain of this receptor accounts for more than 25% of mutations while ~10% mutations are reported in tyrosine kinase domain (FLT3-TKD). AML that initially respond to chemotherapy often relapse and such relapse is more common in AMLs harbouring FLT3 mutations. Although these FLT3 mutations were discovered two decades ago, FLt3/FLT3-ITD elicited signaling and mechanisms underlying disease relapse/chemoresistance in such patients is not clearly understood. Research of our lab focuses to identify downstream kinases involved in FLT3-ITD signaling and how do they inhibit myeloid differentiation.

(d) Drug repurposing for acute myeloid leukemia:

Despite extensive research in myeloid leukemia and improved overall outcome, intensive “7+3” chemotherapy has been main stay of treatment in AML. High cost of discovery and an unmet need for new targeted therapies that are well tolerated, Drug repurposing of existing drugs has emerged as an attractive approach as it involves lesser financial and regulatory hurdles. Our lab also focuses on repurposing of drugs having potential to induce myeloid differentiation in AML cell lines which we further validate in blasts from AML patients.

(e) Role of E3s in invasive breast cancer:

Approximately 10–15% of patients with breast cancer has an aggressive disease and develops distant metastases within 3 years after the initial detection of the primary tumor. The heterogeneous nature of breast cancer metastasis makes it difficult not only to define cure for this disease, but also to assess risk factors for metastasis. Owing to their invasive potential, it is very likely that they manipulate the protein turnover of key regulatory proteins involved in tumor regression. As protein turnover is maintained by E3s and DUBs, we hypothesize that identification deregulated E3s, DUBs and their substrates may be pivotal to understanding the pathophysiology of breast cancer invasion and better therapeutics.

(f) Elucidating downstream signaling of RUNX2 and sclerostin in bone biology:

Runx2 is a master regulator of bone formation that promotes osteoblast differentiation by directly regulating osteoblast specific genes such as osterix and osteopontin. On the other hand, Sclerostin secreted by terminally differentiated osteocytes inhibits bone formation by inhibiting Wnt pathway. Research in our lab focuses to identify interacting protein partners of Runx2 as well Sclerostin along bone formation using mass spectrometry based proteomics approach.