Hima V. Vangapandu

Somatic Mutations, Allele Loss, and DNA Methylation of the Cub and Sushi Multiple Domains 1 (CSMD1) Gene Reveals Association with Early Age of Diagnosis in Colorectal Cancer Patients

Background The Cub and Sushi Multiple Domains 1 (CSMD1) gene, located on the short arm of chromosome 8, codes for a type I transmembrane protein whose function is currently unknown. CSMD1 expression is frequently lost in many epithelial cancers. Our goal was to characterize the relationships between CSMD1 somatic mutations, allele imbalance, DNA methylation, and the clinical characteristics in colorectal cancer patients. Methods We sequenced the CSMD1 coding regions in 54 colorectal tumors using the 454FLX pyrosequencing platform to interrogate 72 amplicons covering the entire coding sequence. We used heterozygous SNP allele ratios at multiple CSMD1 loci to determine allelic balance and infer loss of heterozygosity. Finally, we performed methylation-specific PCR on 76 colorectal tumors to determine DNA methylation status for CSMD1 and known methylation targets ALX4, RUNX3, NEUROG1, and CDKN2A. Results Using 454FLX sequencing and confirming with Sanger sequencing, 16 CSMD1 somatic mutations were identified in 6 of the 54 colorectal tumors (11%). The nonsynonymous to synonymous mutation ratio of the 16 somatic mutations was 15∶1, a ratio significantly higher than the expected 2∶1 ratio (p = 0.014). This ratio indicates a presence of positive selection for mutations in the CSMD1 protein sequence. CSMD1 allelic imbalance was present in 19 of 37 informative cases (56%). Patients with allelic imbalance and CSMD1 mutations were significantly younger (average age, 41 years) than those without somatic mutations (average age, 68 years). The majority of tumors were methylated at one or more CpG loci within the CSMD1 coding sequence, and CSMD1 methylation significantly correlated with two known methylation targets ALX4 and RUNX3. C:G>T:A substitutions were significantly overrepresented (47%), suggesting extensive cytosine methylation predisposing to somatic mutations. Conclusions Deep amplicon sequencing and methylation-specific PCR reveal that CSMD1 alterations can correlate with earlier clinical presentation in colorectal tumors, thus further implicating CSMD1 as a tumor suppressor gene.

Duvelisib: a phosphoinositide-3 kinase δ/γ inhibitor for chronic lymphocytic leukemia

ABSTRACT Introduction: Frontline chemotherapy is successful against chronic lymphocytic leukemia (CLL), but results in untoward toxicity. Further, prognostic factors, cytogenetic anomalies, and compensatory cellular signaling lead to therapy resistance or disease relapse. Therefore, for the past few years, development of targeted therapies is on the rise. PI3K is a major player in the B-cell receptor (BCR) signaling axis, which is critical for the survival and maintenance of B cells. Duvelisib, a PI3K δ/γ dual isoform specific inhibitor that induces apoptosis and reduces cytokine and chemokine levels in vitro, holds promise for CLL. Areas covered: Herein, we review PI3K isoforms and their inhibitors in general, and duvelisib in particular; examine literature on preclinical investigations, pharmacokinetics and clinical studies of duvelisib either as single agent or in combination, for patients with CLL and other lymphoid malignancies. Expert opinion: Duvelisib targets the PI3K δ isoform, which is necessary for cell proliferation and survival, and γ isoform, which is critical for cytokine signaling and pro-inflammatory responses from the microenvironment. In phase I clinical trials, duvelisib as a single agent showed promise for CLL and other lymphoid malignancies. Phase II and III trials of duvelisib alone or in combination with other agents are ongoing.

B cell receptor signaling regulates metabolism in Chronic Lymphocytic Leukemia.

Peripheral blood chronic lymphocytic leukemia (CLL) cells are quiescent but have active transcription and translation processes, suggesting that these lymphocytes are metabolically active. Based on this premise, the metabolic phenotype of CLL lymphocytes was investigated by evaluating the two intracellular ATP-generating pathways. Metabolic flux was assessed by measuring glycolysis as extracellular acidification rate (ECAR) and mitochondrial oxidative phosphorylation as oxygen consumption rate (OCR) and then correlated with prognostic factors. Further, the impact of B-cell receptor signaling (BCR) on metabolism was determined by genetic ablation and pharmacological inhibitors. Compared with proliferative B-cell lines, metabolic fluxes of oxygen and lactate were low in CLL cells. ECAR was consistently low, but OCR varied considerably in human patient samples (n = 45). Higher OCR was associated with poor prognostic factors such as ZAP 70 positivity, unmutated IGHV, high β2M levels, and higher Rai stage. Consistent with the association of ZAP 70 and IGHV unmutated status with active BCR signaling, genetic ablation of BCR mitigated OCR in malignant B cells. Similarly, knocking out PI3Kδ, a critical component of the BCR pathway, decreased OCR and ECAR. In concert, PI3K pathway inhibitors dramatically reduced OCR and ECAR. In harmony with a decline in metabolic activity, the ribonucleotide pools in CLL cells were reduced with duvelisib treatment. Collectively, these data demonstrate that CLL metabolism, especially OCR, is linked to prognostic factors and is curbed by BCR and PI3K pathway inhibition. Implications: This study identifies a relationship between oxidative phosphorylation in CLL and prognostic factors providing a rationale to therapeutically target these processes. Mol Cancer Res; 15(12); 1692–703. ©2017 AACR.

Biological and metabolic effects of IACS-010759, an OxPhos inhibitor, on chronic lymphocytic leukemia cells

Blood cells from patients with chronic lymphocytic leukemia (CLL) are replicationally quiescent but transcriptionally, translationally, and metabolically active. Recently, we demonstrated that oxidative phosphorylation (OxPhos) is a predominant pathway in CLL for energy production and is further augmented in the presence of the stromal microenvironment. Importantly, CLL cells from patients with poor prognostic markers showed increased OxPhos. From these data, we theorized that OxPhos can be targeted to treat CLL. IACS-010759, currently in clinical development, is a small-molecule, orally bioavailable OxPhos inhibitor that targets mitochondrial complex I. Treatment of primary CLL cells with IACS-010759 greatly inhibited OxPhos but caused only minor cell death at 24 and 48 h. In the presence of stroma, the drug successfully inhibited OxPhos and diminished intracellular ribonucleotide pools. However, glycolysis and glucose uptake were induced as compensatory mechanisms. To mitigate the upregulated glycolytic flux, we used 2-deoxy-D-glucose in combination with IACS-010759. This combination reduced both OxPhos and glycolysis and induced cell death. Consistent with these data, low-glucose culture conditions sensitized CLL cells to IACS-010759. Collectively, these data suggest that CLL cells adapt to use a different metabolic pathway when OxPhos is inhibited and that targeting both OxPhos and glycolysis pathways is necessary for biological effect.

Extracellular Flux Assays to Determine Oxidative Phosphorylation and Glycolysis in Chronic Lymphocytic Leukemia Cells

Extracellular flux assays are conducted using seahorse XF96 analyzer. They are used to calculate oxygen consumption rate which is to determine mitochondrial oxidative phosphorylation and extracellular acidification rate which is a measure of glycolysis. Collectively, these assays are used to assess the metabolic phenotype of a cell. Up to four drugs can be loaded and tested in the XF cartridges used in the assay and their effect on cells could be determined. While adherent cell lines are easy to use for this assay, suspension cultures or primary cells are difficult to use. In the following sections, we describe the methodology for this assay for CLL cells in suspension cultures and CLL-stroma cocultures.

Proteomics profiling identifies induction of caveolin-1 in chronic lymphocytic leukemia cells by bone marrow stromal cells

Abstract Chronic lymphocytic leukemia (CLL) is an indolent B-cell malignancy in which cells reside in bone marrow, lymph nodes, and peripheral blood, each of which provides a unique microenvironment. Although the levels of certain proteins are reported to induce, changes in the CLL cell proteome in the presence of bone marrow stromal cells have not been elucidated. Reverse-phase protein array analysis of CLL cells before and 24 h after stromal cell interaction revealed changed levels of proteins that regulate cell cycle, gene transcription, and protein translation. The most hit with respect to both the extent of change in expression level and statistical significance was caveolin-1, which was confirmed with immunoblotting. Caveolin-1 mRNA levels were also upregulated in CLL cells after stromal cell interaction. The induction of caveolin-1 levels was rapid and occurred as early as 1 h. Studies to determine the significance of upregulated caveolin-1 levels in CLL lymphocytes are warranted.

Abstract 3547: B cell receptor signaling regulates cellular metabolism in Chronic Lymphocytic Leukemia

Peripheral blood B cells in chronic lymphocytic leukemia (CLL) are quiescent, yet have an active RNA transcription and protein translation processes, suggesting that these malignant lymphocytes may be metabolically active. However, CLL metabolism and its relationship to disease biology has not been well-explored. In our previous investigation, we reported that compared with proliferative B-cell lines, metabolic fluxes of oxygen and lactate were low in quiescent peripheral blood B lymphocytes from CLL patients (Vangapandu, H.V. et al, AACR 2014). In 45 patient samples tested, glycolysis (extracellular acidification rate, ECAR) was consistently low (1 to 15 mpH/min/5 x 105 lymphocytes), whereas, oxygen consumption rate (OCR) varied considerably (5 to 190 pMoles/min/5 x 105 lymphocytes). Among the prognostic factors, high OCR correlated strongly with ZAP 70 positivity, unmutated IGHV status, greater β2M levels and higher Rai stage. In contrast, glycolytic flux (ECAR) from same patient samples did not associate with prognostic factors. Further, OCR did not vary on the basis of frequently occurring cytogenetic abnormalities, 13q14, 17p, and 11q deletions or trisomy 12. Since, ZAP 70 and IGHV unmutated status are associated with augmented B-cell receptor (BCR) pathway signaling, we tested the impact on OCR after genetic ablation of B-cell receptor (CH2 region of IgM). A CRISPR-Cas9-mediated BCR knockout mitigated OCR in a malignant B cell line without impacting the rate of proliferation. A critical node in the BCR pathway is PI3 Kinase and PI3K isoforms, δ and γ are involved in B-cell malignancies. Consistent with BCR ablation results, knocking out PIK3CD (codes for catalytic subunit δ), dramatically reduced OCR and ECAR. Pharmacological inhibitors of the PI3K pathway, duvelisib (a PI3K δ/γ inhibitor in phase III clinical trials) or idelalisib (FDA approved PI3K δ inhibitor) also decreased OCR. Direct inhibition of AKT with MK-2206 showed similar results. Collectively, these data suggest that CLL cellular metabolism is associated with prognostic factors and linked to BCR signaling pathway. Since, the PI3K inhibitors used in our study are being tested clinically for patients with CLL, investigations on the impact on metabolomics during therapy and translation of results to combination strategies need to be explored. Note: This abstract was not presented at the meeting. Citation Format: Hima V. Vangapandu, Ondrej Havranek, William G. Wierda, Michael J. Keating, Richard Eric Davis, Christine M. Stellrecht, Varsha Gandhi. B cell receptor signaling regulates cellular metabolism in Chronic Lymphocytic Leukemia [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 3547. doi:10.1158/1538-7445.AM2017-3547

Abstract 2658: Stromal microenvironment modulates mitochondrial metabolism in chronic lymphocytic leukemia cells and is abrogated by PI3K δ and γ inhibition

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of replicationally quiescent mature B cells, as they fail to undergo apoptosis. Supporting stromal cells in the microenvironment provide survival advantage to CLL cells. We hypothesized that stromal cells may have an active role in energy metabolism of malignant B-cells in order to cope with their energy demands and is driven by PI3K pathway. We assessed the two major energy generating pathways (glycolysis and mitochondrial Oxidative Phosphorylation (OXPHOS)), in primary CLL cells using Extracellular Flux Analyzer (Seahorse Bioscience), in presence of three different stromal cell lines. Glycolysis measured as ECAR did not show stroma-induced increase, which is commonly upregulated in most cancers. A decrease in glucose uptake in coculture experiments, further suggested that glycolysis was not upregulated in CLL cells (n = 7, p<0.01). Surprisingly, OXPHOS measured as OCR in CLL cells from 29 patients showed a statistically significant increase, (p<0.01) upon coculture and contact with NK-Tert stromal cells. Similar OCR induction was observed in presence of M2-10B4 and HS-5 stromal cell lines. However, no significant changes were observed in mitochondrial mass, membrane potential and ROS, upon co-culture. All four ribonucleotide triphosphate pools (ATP, CTP, GTP, and UTP), showed a 2-fold and statistically significant increase after interacting with stroma cell line for 48hr. Changes in metabolomics and NTP pools were not due to cell growth as stroma cocultures did not impact proliferation index (Ki-67 staining) of CLL cells. In parallel to these changes in metabolomics, stromal microenvironment increased AKT phosphorylation at Thr308 and Ser473 indicating upregulation of PI3K pathway. Consistently, inhibition of PI3K axis by IPI-145 (duvelisib), a PI3K δ and γ inhibitor, currently in phase III trials for CLL, decreased phospho-AKT in CLL samples (n = 6) and dramatically reduced OCR (n = 7) and ECAR in CLL cells in suspension cultures without causing cytotoxicity. Importantly, stroma-mediated increase in OCR in CLL cells was significantly compromised by IPI-145 treatment (n = 5; p<0.05). In parallel, stroma-driven AKT phosphorylation in CLL samples (n = 6) was also reduced with IPI-145. Preliminary data also indicates a decline in NTP pools, upon drug treatment. Collectively, these data suggest that stroma impacts metabolomics in quiescent CLL cells which is in part driven by PI3K/AKT pathway. Citation Format: Hima V. Vangapandu, Kumudha Balakrishnan, Mary L. Ayres, William G. Wierda, Michael J. Keating, Christine M. Stellrecht, Varsha Gandhi. Stromal microenvironment modulates mitochondrial metabolism in chronic lymphocytic leukemia cells and is abrogated by PI3K δ and γ inhibition. [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 2658. doi:10.1158/1538-7445.AM2015-2658

Abstract 4328: Marrow stromal cells promote mitochondrial energy metabolism in primary CLL cells without impacting cellular proliferation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of replicationally quiescent mature B cells, as they fail to undergo apoptosis. Studies show that supporting stromal cells in the microenvironment provide survival advantage to CLL cells by conferring resistance to spontaneous and drug-induced apoptosis. A recent transcriptome characterization showed that compared to normal lymphocytes, genes involved in metabolic pathways were upregulated in CLL cells (Ferreira et al., Genome Res 2013). We hypothesized that stromal cells may have an active role on energy metabolism of malignant B-cells in order to cope with their energy demands. We assessed the major energy generating pathways (glycolysis and mitochondrial OXPHOS), in primary CLL cells using Extracellular Flux Analyzer (Seahorse Bioscience). The oxygen consumption rate (OCR) in CLL cells is surprisingly high compared to the proliferating lymphoma cell lines. Whereas, the glycolytic flux, measured as extracellular acidification rate (ECAR) is significantly low, suggesting that OXPHOS measured as OCR is upregulated in CLL cells; hence we focused on OXPHOS. Increased OCR was not impacted by age, Rai stage, lymphocyte counts, β2M, IgVH mutation status or LDH levels. However, ZAP70 positive (a poor-prognosis marker) samples had higher OCRs (n=30, p =0.002). Compared to males, samples obtained from female patients had higher OCRs (n=33, p = 0.043). Co-culturing with stroma (NK.tert human cell line) for 18-24 hrs had an impact on OCR in CLL; out of 30 samples, 24 showed a significant (p - 0.0005) increase in basal OCR and/or spare respiratory capacity (SRC, biological parameter of mitochondrial respiration). In addition, 11 of 12 CLL samples showed a significant increase in basal OCR, when plated on stroma without prior incubation (p < 0.0001), whereas the SRC did not statistically change (p - 0.9791). Moreover, the differences associated with gender and ZAP70 status were more pronounced when CLL cells were co-cultured on stroma (ZAP70 status, n= 30, p = 0.0013; Gender n=28, p =0.0173). In contrast to OCR, ECAR did not show stroma-induced statistical increase. Preliminary data with murine stroma line (M2-10B4) also showed upregulation of OCR in CLL cells. The stroma-mediated increase in OCR was not due to an increase in proliferation index; as CLL cells on stroma stained negative for Ki67 (marker for cell proliferation). Metabolite analysis by mass spectrometry of 5 CLL sample sets revealed that TCA cycle and gluconeogenesis were among the top 5 hits of pathways upregulated by stromal co-culture. At the molecular level, CLL cells in presence of stroma showed an increased p-AKT and p-ERK levels. Active AKT modulates GLUT4 protein levels, which was expressed in all 5 samples. Collectively, these data suggest that in CLL cells, OXPHOS (measured as OCR) is high and was further induced by stroma. Citation Format: Hima Venkata Vangapandu, Kumudha Balakrishnan, William G. Wierda, Michael J. Keating, Christine M. Stellrecht, Varsha Gandhi. Marrow stromal cells promote mitochondrial energy metabolism in primary CLL cells without impacting cellular proliferation. [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 4328. doi:10.1158/1538-7445.AM2014-4328