Ramzi M. Mohammad

Broad targeting of resistance to apoptosis in cancer

Apoptosis or programmed cell death is natural way of removing aged cells from the body. Most of the anti-cancer therapies trigger apoptosis induction and related cell death networks to eliminate malignant cells. However, in cancer, de-regulated apoptotic signaling, particularly the activation of an anti-apoptotic systems, allows cancer cells to escape this program leading to uncontrolled proliferation resulting in tumor survival, therapeutic resistance and recurrence of cancer. This resistance is a complicated phenomenon that emanates from the interactions of various molecules and signaling pathways. In this comprehensive review we discuss the various factors contributing to apoptosis resistance in cancers. The key resistance targets that are discussed include (1) Bcl-2 and Mcl-1 proteins; (2) autophagy processes; (3) necrosis and necroptosis; (4) heat shock protein signaling; (5) the proteasome pathway; (6) epigenetic mechanisms; and (7) aberrant nuclear export signaling. The shortcomings of current therapeutic modalities are highlighted and a broad spectrum strategy using approaches including (a) gossypol; (b) epigallocatechin-3-gallate; (c) UMI-77 (d) triptolide and (e) selinexor that can be used to overcome cell death resistance is presented. This review provides a roadmap for the design of successful anti-cancer strategies that overcome resistance to apoptosis for better therapeutic outcome in patients with cancer.

Bortezomib-mediated downregulation of S-phase kinase protein-2 (SKP2) causes apoptotic cell death in chronic myelogenous leukemia cells

BackgroundProteasome inhibitors are attractive cancer therapeutic agents because they can regulate apoptosis-related proteins. Bortezomib also known as Velcade®, a proteasome inhibitor that has been approved by the food and drug administration for treatment of patients with multiple myeloma, and many clinical trials are ongoing to examine to the efficacy of bortezomib for the treatment of other malignancies. Bortezomib has been shown to induce apoptosis and inhibit cell growth of many cancer cells. In current study, we determine whether bortezomib induces cell death/apoptosis in CML.MethodsCell viability was measured using MTT assays. Apoptosis was measured by annexin V/PI dual staining and DNA fragmentation assays. Immunoblotting was performed to examine the expression of proteins. Colony assays were performed using methylcellulose.ResultsTreatment of CML cells with bortezomib results in downregulation of S-phase kinase protein 2 (SKP2) and concomitant stabilization of the expression of p27Kip1. Furthermore, knockdown of SKP2 with small interference RNA specific for SKP2 caused accumulation of p27Kip1. CML cells exposed to bortezomib leads to conformational changes in Bax protein, resulting in loss of mitochondrial membrane potential and leakage of cytochrome c to the cytosol. In the cytosol, cytochrome c causes sequential activation of caspase-9, caspase-3, PARP cleavage and apoptosis. Pretreatment of CML cells with a universal inhibitor of caspases, z-VAD-fmk, prevents bortezomib-mediated apoptosis. Our data also demonstrated that bortezomib treatment of CML downregulates the expression of inhibitor of apoptosis proteins. Finally, inhibition of proteasome pathways by bortezomib suppresses colony formation ability of CML cells.ConclusionsAltogether, these findings suggest that bortezomib suppresses the cell proliferation via induction of apoptosis in CML cells by downregulation of SKP2 with concomitant accumulation of p27Kip1, suggesting that proteasomal pathway may form novel therapeutic targets for better management of CML.

Molecular genetic analysis of consanguineous families with primary microcephaly identified pathogenic variants in the ASPM gene

Autosomal recessive primary microcephaly is a rare genetic disorder that is characterized by reduced head circumference and a varying degree of intellectual disability. Genetic studies on consanguineous families with primary microcephaly have identified 15 (MCPH) causative genes that include MCPH1, WDR62, CDK5RAP2, CASC5, ASPM, CENPJ, STIL, CEP135, CEP152, ZNF335, PHC1, CDK6, CENPE, SASS6MFSD2AANKLE2 and CIT (Khan et al.2014; Yamamoto et al.2014; Alakbarzade et al.2015; Morris-Rosendahl and Kaindl 2015; Basit et al.2016). Physiologically, most of these MCPH proteins are involved in cell cycle and its regulation. In the present clinical genetic study, we have present two consanguineous Pakistani families segregating primary microcephaly and intellectual disability. These families were ascertained from the Saraiki ethnic part of Khyber-Pakhtunkhwa province in Pakistan. Whole exome sequencing in one family revealed a novel 1-bp deletion NM_018136.4: c.10013delA (p.Asp3338Valfs*2), while the other family showed a previously reported nonsense mutation NM_018136.4: c.9730C > T (rs199422195 (p.Arg3244*)) in ASPM gene. The novel frame-shift mutation (p.Asp3338Valfs*2) in ASPM presumably truncates the protein synthesis that results in loss of armadillo-type fold domain.

Systems and Network Understanding of Cancer Stem Cells

The notion that tumors arise from a rare population of cells with stem cell characteristics was first proposed more than a century ago when pathologists like Virchow and Cohnheim formulated the hypothesis that cancer results from the activation of embryonic-tissue remnants (Weiss 2000). Since then, advances in different fields have provided support to this original proposal that has led to the increasingly accepted yet controversial “cancer stem cell (CSC)” hypothesis that explains the development of multiple forms of human cancers (Wicha et al. 2006). The first experiments indicating the existence of these cells were performed in animal models in the 1970s where it was concluded that only a low percentage of transplanted murine lymphoma cells formed colonies in the spleen of recipient animals (Park et al. 1971a; Bruce and VAN DER 1963). Likewise, only a minimum number (1 in 100 to 1 in 100,000) of murine myeloma cells were able to form colonies in

An Overview of Proteomics Techniques and its Application as a Tool in Biomarker and Drug Discovery

Introduction: Proteomics technology is extensively used to identify the underlying molecular mechanisms of various diseases. Emergent technologies in proteomics have been used in the biomarker and drug discovery process. Proper use of this technology can enable the understanding of mechanism of drug action; efficacy and toxicity, there by facilitating effective translation of the drug from bench to bedside. Areas covered: The major techniques used in proteomics technology with an application in drug discovery process are discussed. An overview on different kinds of proteomic approaches and their application in various fields of biomarker discovery as well as drug development process has also been presented. Conclusions: Proteomics technology serves as a promising approach by providing unbiased information about protein-protein interactions, post-translational modifications and regulatory mechanisms. Owing to the complexity of the proteomes the technology needs to be complemented with other omics techniques which could revolutionize drug development process. Advanced instrumentation with improved sensitivity, selectivity coupled with efficient proteomics work flows can facilitate comprehensive characterization of various proteomes for potential drug targets

Systems biology approaches in the design of effective miRNA-targeted therapeutics

MicroRNAs (miRNAs) are short non-coding RNAs that are recognized to epigenetically modulate gene expression by weakly binding to the 3′UTR and/or other sites of target mRNAs. A number of miRNAs have been recognized to be aberrantly expressed in cancer. These findings have led researchers to aggressively pursue miRNAs for cancer diagnostic and therapeutics. However, both identification and targeting of miRNAs is not very straight forward because each miRNA can modulate scores of different genes which in-turn can influence exponential number of different targets. In recent years, there has been a consensus that the complexity of miRNAs requires holistic systems biology approaches to tease out the specific targets of each miRNA, which appears to be context dependent. In this chapter we present the recent advancements in systems and network biology and how these and related technologies are aiding in the design of effective miRNA-based therapeutics against cancer.

Abstract 1771: Novel small molecule pak4 allosteric modulators with activity against pancreatic cancer

The p21-activated kinase 4 (PAK4) acts as a key effector of Rho family GTPases downstream of K-Ras and is found over-expressed in most of the available pancreatic cancer (PC) cell lines but not in normal human pancreatic ductal epithelial cells (HPDE). Gene copy number amplification studies in PC patient cohorts has shown amplification of PAK4. Most importantly, RNA interference of PAK4 suppresses PC cell proliferation making PAK4 an attractive therapeutic target within the K-Ras signaling network. Nevertheless, the previously developed PAK4 Type I ATP competitive inhibitor (PF-3758309; tested in non-pancreatic models) was evaluated in a Phase 1 study and showed undesirable pharmacokinetic properties as well as no objective responses and was subsequently discontinued. In order to fill this scientific void, we evaluated a new class of PAK4 allosteric modulators in PC. Using multiple molecular biology techniques we tested the Pak4 modulators9 activities (in the presence and absence of -ve and +ve controls) in a panel of PC cells lines, PAK4 over-expressing Gemcitabine resistant (GEM-R) PC models and highly resistant flow sorted PC stem cells (CSC). CSC9s are triple positive for CD133+CD44+EpCam+ and undergo epithelial-to-mesenchymal transition (EMT). The toxicity and efficacy of these PAK4 modulators were evaluated in sub-cutaneous mouse models of PC. The novel, orally available PAK4 allosteric modulators (KPT-7189, KPT-8752) show anti-proliferative activity against different PC cell lines (AsPC-1, Colo-357, MiaPaCa-2, L3.6pl and HPAC IC50s Citation Format: Asfar S. Azmi, William Senapedis, Yosef Landesman, Erkan Baloglu, Ori Kalid, Jack Wu, Bin Bao, Amro Aboukameel, Sharon Shacham, Michael Kauffman, Ramzi M. Mohammad. Novel small molecule pak4 allosteric modulators with activity against pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1771. doi:10.1158/1538-7445.AM2014-1771

Abstract 3438: Selective inhibitors of Mcl-1 with potent activity against multiple myeloma patient cells and animal xenografts.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC To date most of the studies targeting apoptotic machinery have focused on inhibiting Bcl2 and BclXL proteins that have shown limited clinical utility. This could be in part due to the over-expression of Myeloid Leukemia-1 (Mcl-1) that carries similar anti-apoptotic functions. Our recent evaluations of Multiple Myeloma (MM) patient derived samples have shown consistent over-expression of Mcl-1 suggesting that it is an attractive therapeutic target. We have discovered a novel druggable site in Mcl-1 protein. Using structure based drug design we have developed small molecule inhibitors (SMIs) of Mcl-1 that bind with high affinity to the BH-3 hydrophobic groove of the protein thereby displacing different pro-apoptotic partners such as Bax, Bak, Bok and Bid with activity against MM cells (IC50 5 μM) and anti-tumor activity in animal xenografts. The lead SMI (UMI-77) inhibits growth and induces apoptosis in MM cells. Molecular analysis of MM cells reveals activation of pro-apoptotic factors post UMI-77 treatment. Co-immunoprecipitation studies show that UMI-77 disrupts the Mcl-1-Bax and Mcl-1-Bak interactions. The drug synergizes with standard proteasome inhibitor Velcade (5 nM) and Carfilosimib (1 nM). In patient derived MM cells (n=45), UMI-77 inhibits growth and induces apoptosis that is concurrent with induction of Bax, Bak and enhancement in cleaved fractions of caspases 3 and 9. At similar concentrations UMI-77 does not affect normal peripheral lymphocytes. UMI-77 is well tolerated by animals at MTD (60 mg/kg i.v.). In a mice lymphoma model, oral administration of UMI-77 significantly reduced tumor growth and histological examination of tumors revealed suppression of Mcl-1 protein expression along with enhancement of pro-apoptotic markers. Our novel Mcl-1 inhibitors provide a new therapeutic option for cancers for MM that overexpress Mcl-1 that have failed standard therapies. Citation Format: Asfar S. Azmi, Zaneta Nikolovka-Coleska, Muneer Abidi, Kelley Marsack, Ashiq Masood, Hiroshi Yano, Silvana Pregja, Jeffrey Zonder, Ramzi M. Mohammad. Selective inhibitors of Mcl-1 with potent activity against multiple myeloma patient cells and animal xenografts. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3438. doi:10.1158/1538-7445.AM2013-3438

Abstract 3445: Specific inhibitors of nuclear export (SINE) for cancer therapy: from bench to bedside.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Nuclear protein transport is an evolutionary conserved mechanism that controls aberrant cell proliferation through balanced nuclear expression of tumor suppressor proteins (TSPs). In cancers, overexpression of exportin protein chromosome region maintenance 1 (CRM-1) disrupts this balance leading to nuclear exclusion of TSPs leading to the low efficacy of traditional anti-cancer drugs. Our recent screening of pancreatic, colon, triple negative breast cancer (TNBC) and non-Hodgkins lymphoma (NHL) patient samples showed over-expression of CRM-1. These important finding suggests that therapeutic targeting of CRM-1 leading to restoration of TSPs in the nucleus is an attractive strategy against cancer. Earlier attempts to develop CRM-1 inhibitor were not successful as exemplified by the failure of the Leptomycin B in a single clinical trial due to severe off-target toxicity. Since then the field has not witnessed any serious attempts to develop newer classes of CRM-1 inhibitors that could be used clinically. We have developed specific inhibitors of nuclear export (SINE) that are highly specific, orally active drugs with excellent pharmacokinetic parameters. We tested SINEs as targeted inhibitors of CRM-1, in a panel of pancreatic, colon, prostate, breast, NHL cell lines. SINE showed cancer selectivity (IC50s in low nano molar range in cancer cells and >20 micro Molar in normal lymphocytes, fibroblasts and NIH-3T3 cells). SINE induces global re-alignment of important tumor suppressors such as IKB, FOXO, p53, p73 and p27. SINEs were ineffective in killing cancer cells that were transduced with cys528 mut-CRM-1 proteins (a binding site for SINE) confirming the drugs selectivity. In in vivo studies, the clinical grade SINE induced significant tumor growth inhibition in subcutaneous and orthotopic solid and disseminated NHL xenografts. Our new class of SINEs has excellent pharmacokinetic parameters and has passed advanced toxicity profiling. Based on our strong preclinical data, SINE has entered clinical trials for both solid tumors and hematological malignancies. Citation Format: Asfar S. Azmi, Bin Bao, Michael Kauffman, Sharon Shacham, Ramzi M. Mohammad. Specific inhibitors of nuclear export (SINE) for cancer therapy: from bench to bedside. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3445. doi:10.1158/1538-7445.AM2013-3445

Abstract 5234: Network interrogations on SNAIL inhibitor GN-25 induced perturbations in HMLE-SNAIL cell line models.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC HMLEs (HMLE-SNAIL and Kras-HMLE-SNAIL pairs) serve as excellent model system to interrogate the effect of SNAIL targeted agents that reverse epithelial-to-mesenchymal transition (EMT). We developed a SNAIL-p53 interaction inhibitor (GN-25) that was shown to suppress SNAIL function. In this report, using pathway network analysis, we show that GN-25 induces the reversal of EMT to mesenchymal-to-epithelial transition (MET) in well recognized HMLE-SNAIL and Kras-HMLE-SNAIL models. GN-25 induced MET was found to be concurrent with growth inhibition, suppression of spheroid forming capacity and induction of apoptosis. We performed systems and pathway network analysis on mRNA expression by microarrays from GN-25 treated Kras-HMLE-SNAIL cells that showed an orchestrated global re-organization of EMT network genes. The expression signatures were validated at the protein level using confocal microscopy, western blot analysis and time lapse videography, and we found down-regulation of mesenchymal markers such as TWIST1, TWIST2, ZEB-1 concurrently with up-regulation of epithelial marker E-Cadherin. Additionally, RNAi studies validated SNAIL dependent mechanism of action of the drug. Most importantly, GN-25 deregulated many major transcription factors (TFs) such as inhibition of oncogenic TFs Myc, TBX2, NR3C1 and enhancement in tumor suppressor TFs such as SMAD7, DD1T3, CEBPA, HOXA5, TFEB, IRF1, IRF7 and XBP1, resulting in MET as well as induction of cell death. Our systems and network investigations provide solid pre-clinical supportive evidence for clinical application of GN-25 for the reversal of EMT phenotype to MET in aggressive cancers. Citation Format: Asfar S. Azmi, Aliccia Bollig-Fisher, Bin Bao, Bum Joon-Park, SH Lee, Gyu-Yong Song, Ramzi M. Mohammad, Fazlul H. Sarkar. Network interrogations on SNAIL inhibitor GN-25 induced perturbations in HMLE-SNAIL cell line models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5234. doi:10.1158/1538-7445.AM2013-5234