Rekha Pal

Use of microsatellite marker loss of heterozygosity in accurate diagnosis of pancreaticobiliary malignancy from brush cytology samples

Background: Brush cytology of biliary strictures to diagnose pancreaticobiliary malignancy suffers from poor sensitivity. Aim: To improve the diagnostic yield of pancreaticobiliary brush cytology through analysis of tumour suppressor gene linked microsatellite marker loss of heterozygosity (LOH) and k-ras codon 12 mutation detection. Methods: Twenty six patients with biliary strictures underwent endoscopic retrograde cholangiography with brush cytology. A panel of 12 polymorphic microsatellite markers linked to six tumour suppressor genes was developed. Genomic DNA from cell clusters acquired from brush cytology specimens and microdissected surgical malignant and normal tissue underwent polymerase chain amplification reaction (PCR). PCR products were compared for LOH and k-ras codon 12 mutations. Results: Seventeen patients were confirmed to have pancreaticobiliary adenocarcinoma. Nine patients had benign strictures (eight proven surgically, one by follow up). Cytomorphological interpretation was positive for malignancy (n = 8), indeterminate (n = 10), and negative for malignancy (n = 8). Selected malignant appearing cytological cell clusters and microdissected histological samples from cancer showed abundant LOH characteristic of malignancy while brushings from nine cases without cancer carried no LOH (p<0.001). LOH and k-ras mutations profile of the cytological specimens was almost always concordant with the tissue samples. Presence of k-ras mutation predicted malignancy of pancreatic origin (p<0.001). Conclusion: LOH and k-ras codon 12 mutation analysis of PCR amplified DNA from biliary brush cytology discriminates reactive from malignant cells, with 100% sensitivity, specificity, and accuracy. Minor variations in LOH in brushings and in different sites within the same tumour likely reflect intratumoral mutational heterogeneity during clonal expansion of pre- and neoplastic lineages.

IMiD immunomodulatory compounds block C/EBPβ translation through eIF4E down-regulation resulting in inhibition of MM

Immunomodulatory derivatives of thalidomide (IMiD compounds), such as pomalidomide and lenalidomide, are highly active in multiple myeloma (MM) treatment. However, the precise mechanisms of action and resistance in MM are unresolved. Here we show that IMiD compounds down-regulate CCAAT/enhancer-binding protein-β (C/EBPβ) resulting in abrogation of cell proliferation. Overexpression of C/EBPβ rescued MM cells from IMiD-induced inhibition of proliferation, indicating that C/EBPβ is critical in mediating antiproliferative effects. IMiD-induced decrease of C/EBPβ protein led to impaired transcription of interferon regulatory factor 4 (IRF4). Down-regulation of IRF4 by lenalidomide was confirmed by longitudinal studies of bone marrow samples from 23 patients obtained before and during lenalidomide treatment using CD138⁺/IRF4⁺ double labeling. In contrast to down-regulation of C/EBPβ protein, IMiD compounds did not alter C/EBPβ mRNA levels or protein stability, suggesting translational regulation of C/EBPβ. We could demonstrate that C/EBPβ protein expression is under eIF4E-translational control in MM. Furthermore, inhibition of the eIF4E-C/EBPβ axis by IMiD compounds was not observed in IMiD-resistant MM cells. However, targeting translation at a different level by inhibiting eukaryotic translation initiation factor 4E-binding protein 1 phosphorylation overcame resistance, suggesting that this pathway is critical and might be a target to overcome drug resistance.

Immunomodulatory derivatives induce PU.1 down-regulation, myeloid maturation arrest, and neutropenia

The immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide yield high response rates in patients with multiple myeloma, but the use of IMiDs in multiple myeloma is associated with neutropenia and increased risk for venous thromboembolism (VTE) by mechanisms that are unknown. We show that IMiDs down-regulate PU.1, a key transcription factor involved in granulocyte differentiation in vitro and in patients treated with lenalidomide. Loss of PU.1 results in transient maturation arrest with medullary accumulation of immature myeloid precursors and subsequent neutropenia. Accumulation of promyelocytes leads to high levels of the platelet aggregation agonist, cathepsin G stored in the azurophilic granules of promyelocytes. High levels of cathepsin G subsequently may increase the risk of VTE. To our knowledge, this is the first report investigating the underlying mechanism of IMiD-induced neutropenia and increased risk of VTE in multiple myeloma.

C/EBPβ regulates transcription factors critical for proliferation and survival of multiple myeloma cells

CCAAT/enhancer-binding protein beta (C/EBPbeta), also known as nuclear factor-interleukin-6 (NF-IL6), is a transcription factor that plays an important role in the regulation of growth and differentiation of myeloid and lymphoid cells. Mice deficient in C/EBPbeta show impaired generation of B lymphocytes. We show that C/EBPbeta regulates transcription factors critical for proliferation and survival in multiple myeloma. Multiple myeloma cell lines and primary multiple myeloma cells strongly expressed C/EBPbeta, whereas normal B cells and plasma cells had little or no detectable levels of C/EBPbeta. Silencing of C/EBPbeta led to down-regulation of transcription factors such as IRF4, XBP1, and BLIMP1 accompanied by a strong inhibition of proliferation. Further, silencing of C/EBPbeta led to a complete down-regulation of antiapoptotic B-cell lymphoma 2 (BCL2) expression. In chromatin immunoprecipitation assays, C/EBPbeta directly bound to the promoter region of IRF4, BLIMP1, and BCL2. Our data indicate that C/EBPbeta is involved in the regulatory network of transcription factors that are critical for plasma cell differentiation and survival. Targeting C/EBPbeta may provide a novel therapeutic strategy in the treatment of multiple myeloma.

An Opposite Effect of the CDK Inhibitor, p18INK4c on Embryonic Stem Cells Compared with Tumor and Adult Stem Cells

Self-renewal is a feature common to both adult and embryonic stem (ES) cells, as well as tumor stem cells (TSCs). The cyclin-dependent kinase inhibitor, p18INK4c, is a known tumor suppressor that can inhibit self-renewal of tumor cells or adult stem cells. Here, we demonstrate an opposite effect of p18 on ES cells in comparison with teratoma cells. Our results unexpectedly showed that overexpression of p18 accelerated the growth of mouse ES cells and embryonic bodies (EB); on the contrary, inhibited the growth of late stage teratoma. Up-regulation of ES cell markers (i.e., Oct4, Nanog, Sox2, and Rex1) were detected in both ES and EB cells, while concomitant down-regulation of various differentiation markers was observed in EB cells. These results demonstrate that p18 has an opposite effect on ES cells as compared with tumor cells and adult stem cells. Mechanistically, expression of CDK4 was significantly increased with overexpression of p18 in ES cells, likely leading to a release of CDK2 from the inhibition by p21 and p27. As a result, self-renewal of ES cells was enhanced. Our current study suggests that targeting p18 in different cell types may yield different outcomes, thereby having implications for therapeutic manipulations of cell cycle machinery in stem cells.

microRNA-10b Is Overexpressed and Critical for Cell Survival and Proliferation in Medulloblastoma.

This study demonstrates the effects of miRNA-10b on medulloblastoma proliferation through transcriptional induction of the anti-apoptotic protein BCL2. Using a cancer specific miRNA-array, high expression of miRNA-10b in medulloblastoma cell lines compared to a normal cerebellar control was shown, and this was confirmed with real time PCR (RT-PCR). Two medulloblastoma cell lines (DAOY and UW228) were transiently transfected with control miRNA, miRNA-10b inhibitor or miRNA-10b mimic and subjected to RT-PCR, MTT, apoptosis, clonogenic assay and western blot analysis. Transfection of miRNA-10b inhibitor induced a significant down-regulation of miRNA-10b expression, inhibited proliferation, and induced apoptosis, while miRNA-10b mimic exerted an opposite effect. Inhibition of miRNA-10b abrogated the colony-forming capability of medulloblastoma cells, and markedly down-regulated the expression of BCL2. Down-regulation of BCL2 by antisense oligonucleotides or siRNA also significantly down-regulated miRNA-10b, suggesting that BCL2 is a major mediator of the effects of miRNA-10b. ABT-737 and ABT-199, potent inhibitors of BCL2, downregulated the expression of miRNA-10b and increased apoptosis. Analysis of miRNA-10b levels in 13 primary medulloblastoma samples revealed that the 2 patients with the highest levels of miRNA-10b had multiple recurrences (4.5) and died within 8 years of diagnosis, compared with the 11 patients with low levels of miRNA-10b who had a mean of 1.2 recurrences and nearly 40% long-term survival. The data presented here indicate that miRNA-10b may act as an oncomir in medulloblastoma tumorigenesis, and reveal a previously unreported mechanism with Bcl-2 as a mediator of the effects of miRNA-10b upon medulloblastoma cell survival.

Molecular subtypes of colorectal cancer in pre-clinical models show differential response to targeted therapies: Treatment implications beyond KRAS mutations

Molecular subtypes of colorectal tumors are associated with prognosis and prediction for treatment benefit from chemotherapy. The purpose of this study was two-fold: 1) to determine the association of colorectal (CRC) molecular subtypes with response to targeted therapies in pre-clinical models and 2) to identify treatments for CRC stem-like subtype because these tumors are associated with a very poor patient prognosis. Eleven CRC cell lines were classified into molecular subtypes and tested for their response to pan-ERBB, MEK, and ERK inhibitors as single agents and in combination. All six inflammatory or TA cell lines were exquisitely sensitive to the combination of MEK and neratinib whereas all five stem-like cell lines were resistant. Growth inhibition in sensitive cell lines was greater with the combination than with either drug alone even in cell lines with KRAS mutations. The combination inhibited pERK in inflammatory cell lines but not in four out of five stem-like cell lines. MEK162 plus neratinib were synergistic in cell culture and xenograft models in inflammatory cell lines. The ERK inhibitor, SCH772984, down-regulated pERK, decreased cell viability, and was synergistic with neratinib in both inflammatory and stem-like subtypes. These results suggest that inhibition of pERK is a critical node in decreasing cell viability of stem-like CRC tumors. Our results also suggest that CRC molecular subtypes may yield predictive information and may help to identify patients who may respond to targeted inhibitors.

Abstract 5167: Stem-like colorectal cancer cell lines show response to the ERK1/2 inhibitor, SCH772984, alone and in combination with neratinib

Background: We have recently shown an association of colorectal tumor subtypes with differential response to chemotherapy in patients (pts), and to targeted therapy in cell lines. Pts enrolled in NSABP/NRG C-07 with stem-like tumors had a poor prognosis regardless of stage or treatment, highlighting the importance of finding new treatments for stem-like tumors. Our in-vitro data showed that KRAS mutant (mt) cell lines of the intrinsic inflammatory subtype were sensitive to the combination of MEK-162 and neratinib, but stem-like subtype cell lines were resistant regardless of KRAS mt status. The purpose of this study was to extend our observations to xenograft models and to identify new agents that target the stem-like subtype. Methods: KRAS mt cell lines of the inflammatory (NCI-H747) or stem-like (SW-480) subtype were used in xenograft models to test inhibition of tumor growth by MEK-162 and neratinib, alone or in combination. Five stem-like cell lines with KRAS wt (C2BBE1, HS675T) or KRAS mt (SW480, SW620, HCT116), and two KRAS mt inflammatory cell lines (NCI-H747, SW837) were tested for cell viability after treatment with SCH772984 alone or in combination with neratinib. Results: In vivo analysis showed that treatment of NCI-H747 xenografts with a combination of MEK-162 (3mg/kg) and neratinib (10mg/kg) led to significant tumor regression compared to treatment with either drug alone (p ≥0.0001 v neratinib alone; p ≥0.002 v MEK-162 alone). In contrast, MEK-162 alone was able to inhibit tumor growth of stem-like cell line (SW480) xenografts (p ≥0.0061 v control) but neratinib was ineffective (p ≥0.145 v control) as a single agent and did not add to MEK-162. Our in-vitro and in-vivo data showed that inhibition of p-ERK directly correlated with sensitivity to the MEK and neratinib combination in inflammatory subtype. SCH772984 significantly inhibited cell viability of both inflammatory (IC50 1-2 μM) and stem-like subtype (IC50 1-2 μM) cell lines. The combination of neratinib (0.125 μM) and SCH772984 (1 μM) was effective at decreasing cell viability by 60-70% in both inflammatory and stem-like cell lines. Inhibition of ERK phosphorylation was correlated with loss of cell viability. Conclusion: The combination of MEK-162 and neratinib work synergistically to decrease tumor growth in inflammatory but not stem-like colorectal cancer subtypes. We demonstrate that SCH772984 decreases the viability of stem-like colon cancer cell lines, and works synergistically with neratinib. The use of SCH772984 alone, but more potently in combination with neratinib, may represent a therapeutic approach for pts with stem-like tumors, a finding that underscores the potential importance of employing subtype analysis in the diagnosis of colon cancer and potentially as to a guide to new therapies. Support: PA DoH, which disclaims certain responsibility. Citation Format: Rekha Pal, Ning Wei, Nan Song, Shao-yu Wu, S. Rim Kim, Patrick G. Gavin, Peter C. Lucas, Ashok Srinivasan, Samuel A. Jacobs, Soonmyung Paik, John C. Schmitz, Kay Pogue-Geile. Stem-like colorectal cancer cell lines show response to the ERK1/2 inhibitor, SCH772984, alone and in combination with neratinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5167. doi:10.1158/1538-7445.AM2017-5167

Abstract 706: Inflammatory and stem-like colorectal cell lines show differential response to MEK-162 and neratinib in combination

Background: Improvement of colorectal cancer (CRC) treatment depends on finding new effective agents and associated biomarkers that can predict patient response to drugs. Recent cell culture experiments show that a combination of MEK and pan ERBB inhibitors may be efficacious in treating CRC (Sun et al 2014). The purpose of this study was to determine whether a cell line9s response to targeted agents would correlate with its mutation profile and/or gene expression subtype. Methods: K-RAS mutation status of 7 cell lines were identified and classified into stem-like or inflammatory subtypes using gene expression data from the cancer cell line encyclopedia and an algorithm described by Sadanandam et al. We tested 5 stem-like cell lines (SW480, SW620, HCT116, C2BBE1, HS675T) and 2 inflammatory cell lines (NCI-H747, SW837) for response to inhibitors of the RAS/RAF and PI3K pathways. MEK-162 and neratinib were tested as single agents and in combination, using cell viability and clonogenic assays. Results: Cell lines varied in response to the drugs when used as single agents. All cell lines were resistant to the EGFR inhibitor (Gefitinib) at concentrations ≥5 μM. Two pan ERBB inhibitors (neratinib and afatinib) showed IC50 values at or >4 μM and 2 MEK inhibitors (Selumetinib and MEK162) had IC50 values at or >1μM. The inflammatory cell lines, SW837 and NCI-H747, had greater sensitivity to the MEK inhibitors (IC50 value 1μM) while the KRAS mutant stem-like cell lines were relatively more resistant (SW480, SW620) with IC50 values ≥4-5 μM. Stem-like cell lines with WT-KRAS (C2BBE1, HS675T) were even more resistant; IC50s were not achieved at concentrations of 5 μM. Similar results were obtained in clonogenic growth assay. The MEK and pan ERBB inhibitor combinations were more effective at decreasing cell viability and inducing apoptosis in inflammatory cell lines than in stem-like cell lines. This combination effectively inhibited 60-70% of cell viability in inflammatory cell lines at a dose of 0.5 μM and 0.062 μM for neratinib and MEK-162 respectively. The lethality of this combination was the same even if the doses were reversed (0.5 μM MEK-162 and 0.062 μM neratinib). In contrast, we observed that this combination was not nearly as effective against stem-like cell lines irrespective of KRAS mutation status. Profiling of the phosphorylation status of proteins in the RAF-MEK-ERK signaling pathway after exposure to the MEK-162 plus neratinib combination showed a substantial inhibition of ERK phosphorylation in inflammatory cell lines but not in 4 of the 5 stem-like cell lines. Conclusion: This study demonstrates that colon cancer cell lines classified as the inflammatory subtype are sensitive to the combination of MEK-162 plus neratinib at concentrations at which cell lines of the stem-like subtype are resistant. The PA DOH disclaims responsibility for analysis, interpretations, or conclusions. Citation Format: Rekha Pal, Nan Song, Ashok Srinivasan, Samuel A. Jacobs, Soonmyung Paik, Katherine L. Pogue-Geile. Inflammatory and stem-like colorectal cell lines show differential response to MEK-162 and neratinib in combination. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 706. doi:10.1158/1538-7445.AM2015-706