Linda B. Baughn

Profiling Bortezomib Resistance Identifies Secondary Therapies in a Mouse Myeloma Model

Multiple myeloma is a hematologic malignancy characterized by the proliferation of neoplastic plasma cells in the bone marrow. Although the first-to-market proteasome inhibitor bortezomib (Velcade) has been successfully used to treat patients with myeloma, drug resistance remains an emerging problem. In this study, we identify signatures of bortezomib sensitivity and resistance by gene expression profiling (GEP) using pairs of bortezomib-sensitive (BzS) and bortezomib-resistant (BzR) cell lines created from the Bcl-XL/Myc double-transgenic mouse model of multiple myeloma. Notably, these BzR cell lines show cross-resistance to the next-generation proteasome inhibitors, MLN2238 and carfilzomib (Kyprolis) but not to other antimyeloma drugs. We further characterized the response to bortezomib using the Connectivity Map database, revealing a differential response between these cell lines to histone deacetylase (HDAC) inhibitors. Furthermore, in vivo experiments using the HDAC inhibitor panobinostat confirmed that the predicted responder showed increased sensitivity to HDAC inhibitors in the BzR line. These findings show that GEP may be used to document bortezomib resistance in myeloma cells and predict individual sensitivity to other drug classes. Finally, these data reveal complex heterogeneity within multiple myeloma and suggest that resistance to one drug class reprograms resistant clones for increased sensitivity to a distinct class of drugs. This study represents an important next step in translating pharmacogenomic profiling and may be useful for understanding personalized pharmacotherapy for patients with multiple myeloma. Mol Cancer Ther; 12(6); 1140–50. ©2013 AACR.

Reduced CXCR4 expression is associated with extramedullary disease in a mouse model of myeloma and predicts poor survival in multiple myeloma patients treated with bortezomib

Drug resistance to the proteasome inhibitor, bortezomib/VELCADE (Bz) is a significant clinical problem in the treatment of multiple myeloma (MM), an invariably fatal plasma cell malignancy of the bone marrow.1 Despite initial success and wide use in cocktail regimens for MM treatment,2 the majority of patients treated with Bz eventually relapse, many having developed ‘acquired’ Bz-resistant (BzR) disease.1 Thus, the elucidation of mechanisms by which Bz resistance may occur and the identification of novel intervention strategies are important translational aims. To identify mechanisms of acquired Bz resistance, we previously utilized in vitro cell lines from the Bcl-XL/Myc mouse model of plasma cell malignancy to systematically ascertain differences between Bz-sensitive (BzS) and derived BzR cells.3 We have employed this model system because malignant plasma cell lines isolated from these mice closely resemble human MM based on gene expression, chromosomal abnormalities and progression of disease in the bone marrow.3–5 Perhaps most importantly, from initially drug-sensitive tumor cell populations we are able to select for drug-resistant cells in vitro, adoptively transfer these cells back into syngeneic recipient mice, and recapitulate the drug-sensitive or -resistant phenotype following in vivo Bz treatment of recipient mice.3 Remarkably, the differences in Bz sensitivity in our mouse cell lines strongly correlated with differences in malignant plasma cell migration following adoptive transfer. BzR cells displayed a significantly reduced affinity for the bone marrow compartment, compared with their sensitive counterparts, and instead infiltrated extramedullary tissues more readily,3 suggesting the possibility that BzR plasma cells (PCs) are more likely to reside outside of the bone marrow milieu. Thus, we hypothesized that Bz treatment and acquisition of resistance selects for cells that can survive independent of the bone marrow microenvironment and that this phenotype may be due to the loss of cell-surface proteins that mediate the MM–bone marrow stromal cell (BMSC) interaction. We recently identified a 23-gene signature that distinguishes between BzS and BzR mouse cell lines in vitro and significantly predicts differences in patient outcomes in a human MM clinical trial that included Bz.3 We explored the gene expression profiling (GEP) data sets associated with these studies further, and of the 23 genes in this model, 5 genes including chemokine (C-X-C motif) receptor 4 (CXCR4), regulator of G-protein signaling 16 (RGS16), lectin, gaglactoside-binding, soluble, 1 (LGALS1), CD93 and cystathionine-beta-synthase (CBS) have been associated with the trafficking of cells within the immune system (Ingenuity Pathway Analysis). However, of these genes, CXCR4 has been most directly associated with PC migration in both humans and mice.6 We observed a loss in Cxcr4 mRNA expression in all four cases of BzR compared with BzS mouse cell lines.3 CXCR4 is known to be highly expressed on the surface of human plasma cells and contributes to bone marrow homing during normal plasma cell maturation through interaction with its ligand SDF-1α that is secreted by bone marrow stromal cells,6 making this gene an attractive candidate for our hypothesis. Therefore, we began by validating the protein expression of CXCR4 by flow cytometry in BzS and their derived BzR counterpart mouse cell lines we had isolated as previously described.3 The 2.5-fold average reduction in Cxcr4 mRNA expression that we observed by gene expression microarray3 was further reflected as a 1.5- (cell line 589) to 2 (cell line 595)-fold reduction in total (intracellular and surface) CXCR4 protein in the mouse BzR cell lines in vitro (Figure 1a), suggesting that this could be a functional change in the transcriptional regulation of Cxcr4 or that Bz selects for those cells that express lower levels of CXCR4. However, given that the original mRNA expression data were collected in the absence of cell death over a short-time period3 suggests the possibility that Bz contributes to the reduction of Cxcr4 mRNA expression. Figure 1 Low CXCR4 expression is associated with bortezomib-resistance and poor clinical outcomes in patients treated with bortezomib The reduction in CXCR4 expression in BzR cells combined with the in vivo data showing that BzR cells promote aggressive, extramedullary disease resulting in poorer survival3 suggests that CXCR4 could serve as a biomarker for patient survival. We next sought to determine whether reduced CXCR4 expression was associated with poorer survival in MM patients treated with Bz. The APEX drug trial provides initial GEP data from MM patients treated with either single-agent Bz or high-dose dexamethasone.7 We divided the data set into those patients with high versus low CXCR4 expression and assessed overall survival (OS) trends in both groups. As a single biomarker, low CXCR4 expression significantly distinguished those MM patients with poorer OS (P=0.039) in the Bz-treated arm of the clinical trial (Figure 1b), but not the dexamethasone arm (data not shown), suggesting that this could be a novel biomarker associated with poor response to Bz. Because the majority of MM patients are placed on a therapeutic cocktail of drugs that often includes Bz, we next queried the MM total therapy 3 (MMTT3) drug trial that reported GEP data for MM patients treated with VTD-PACE (Bz, thalidomide, dexamethasone, cisplatin, doxorubicin, cyclophosphamide, etoposide) in conjunction with tandem transplantation.8,9 As with the APEX drug trial, low CXCR4 expression significantly distinguished those MM patients with poorer event-free (P=0.013; left panel) and OS (P=0.010; right panel) survival (Figure 1c). Therefore, reduced CXCR4 expression predicts poorer survival in patients treated with Bz. Using the Bcl-XL/Myc model system of plasma cell malignancy, we identified CXCR4 as a single biomarker whose reduced expression is associated with poorer outcomes in MM patients being treated with Bz. CXCR4 expression is known to have a role in PC homing to the bone marrow.10 When injected back into syngeneic mice, BzR cells, which have reduced CXCR4 expression, display reduced BM homing compared with their BzS counterparts, resulting in a much more severe disease phenotype.3 This finding suggests that reduced CXCR4 expression correlates with increased disease severity and agrees with our analysis of both a single-agent Bz clinical trial (APEX7) and a clinical trial utilizing Bz in cocktail with additional agents (MMTT38), both of which show that low CXCR4 expression is significantly associated with worse outcome compared to patients with high CXCR4 expression. The biological role of CXCR4 is of particular interest given that studies have shown that the disruption of the interaction between BMSCs and MM cells (for example, by virtue of chemical inhibition of the CXCR4/SDF-1α axis) may increase sensitivity to Bz by mobilizing MM cells from the bone marrow microenvironment. 11 While the full mechanism of reduced CXCR4 expression in BzR cells awaits further investigation, we have evidence to suggest that Bz treatment promotes the reduction of CXCR4 mRNA, which we have previously reported.3 In turn, over time following continuous Bz treatment, reduced Cxcr4 expression may decrease the reliance of the MM cells on the bone marrow microenvironment and thus eventually promote extramedullary disease. Indeed, BzR disease presenting in patients as extramedullary masses has been reported.12 A circulating malignant clonal PC population has already been described by others as highly drug resistant and likely to contribute to disease relapse.13 Whether this population contributes to the proposed tumor-initiating and drug resistant MM ‘side population’,14 which also expresses lower CXCR4 transcript than the bulk tumor population (FZ, unpublished data), will require additional studies. In summary, these results support the use of CXCR4 as a valuable diagnostic biomarker that predicts clinical outcome in MM patients treated with Bz. The positive impact of cell-surface markers that may predict Bz response as a diagnostic tool in patient care could mean the early detection of BzR disease and improved OS through individualized medical treatment and new therapeutic targets (Supplementary Information).

CDK2 Phosphorylation of Smad2 Disrupts TGF-β Transcriptional Regulation in Resistant Primary Bone Marrow Myeloma Cells

Resistance to growth suppression by TGF-β1 is common in cancer; however, mutations in this pathway are rare in hematopoietic malignancies. In multiple myeloma, a fatal cancer of plasma cells, malignant cells accumulate in the TGF-β-rich bone marrow due to loss of both cell cycle and apoptotic controls. Herein we show that TGF-β activates Smad2 but fails to induce cell cycle arrest or apoptosis in primary bone marrow myeloma and human myeloma cell lines due to its inability to activate G1 cyclin-dependent kinase (CDK) inhibitors (p15INK4b, p21CIP1/WAF1, p27KIP1, p57KIP2) or to repress c-myc and Bcl-2 transcription. Correlating with aberrant activation of CDKs, CDK-dependent phosphorylation of Smad2 on Thr8 (pT8), a modification linked to impaired Smad activity, is elevated in primary bone marrow myeloma cells, even in asymptomatic monoclonal gammopathy of undetermined significance. Moreover, CDK2 is the predominant CDK that phosphorylates Smad2 on T8 in myeloma cells, leading to inhibition of Smad2-Smad4 association that precludes transcriptional regulation by Smad2. Our findings provide the first direct evidence that pT8 Smad2 couples dysregulation of CDK2 to TGF-β resistance in primary cancer cells, and they suggest that disruption of Smad2 function by CDK2 phosphorylation acts as a mechanism for TGF-β resistance in multiple myeloma.

Integration of cytogenomic data for furthering the characterization of pediatric B-cell acute lymphoblastic leukemia: A multi-institution, multi-platform microarray study

It is well documented that among subgroups of B-cell acute lymphoblastic leukemia (B-ALL), the genetic profile of the leukemic blasts has significant impact on prognosis and stratification for therapy. Recent studies have documented the power of microarrays to screen genome-wide for copy number aberrations (CNAs) and regions of copy number-neutral loss of heterozygosity (CNLOH) that are not detectable by G-banding or fluorescence in situ hybridization (FISH). These studies have involved application of a single array platform for the respective cases. The present investigation demonstrates the feasibility and usefulness of integrating array results from multiple laboratories (ARUP, The Childrens Hospital of Philadelphia, Cincinnati Childrens Hospital Medical Center, and University of Minnesota Medical Center) that utilize different array platforms (Affymetrix, Agilent, or Illumina) in their respective clinical settings. A total of 65 patients enrolled on the Childrens Oncology Group (COG) study AALL08B1 were identified for study, as cytogenetic and FISH studies had also been performed on these patients, with a central review of those results available for comparison. Microarray data were first analyzed by the individual laboratories with their respective software systems; raw data files were then centrally validated using NEXUS software. The results demonstrated the added value of integrating multi-platform data with cytogenetic and FISH data and highlight novel findings identified by array including the co-occurrence of low and high risk abnormalities not previously reported to coexist within a clone, novel regions of chromosomal amplification, clones characterized by numerous whole chromosome LOH that do not meet criteria for doubling of a near-haploid, and characterization of array profiles associated with an IKZF1 deletion. Each of these findings raises questions that are clinically relevant to risk stratification.

Utilization of translational bioinformatics to identify novel biomarkers of bortezomib resistance in multiple myeloma.

Multiple myeloma (MM) is an incurable malignant neoplasm hallmarked by a clonal expansion of plasma cells, the presence of a monoclonal protein in the serum and/or urine (M-spike), lytic bone lesions, and end organ damage. Clinical outcomes for patients with MM have improved greatly over the last decade as a result of the re-purposing of compounds such as thalidomide derivatives, as well as the development of novel chemotherapeutic agents including first and second generation proteasome inhibitors, bortezomib (Bz) and carfilzomib. Unfortunately, despite these improvements, the majority of patients relapse following treatment. While Bz, one of the most commonly used proteasome inhibitors, has been successfully incorporated into clinical practice, some MM patients have de novo resistance to Bz, and the majority of the remainder subsequently develop drug resistance following treatment. A significant gap in clinical care is the lack of a reliable clinical test that would predict which MM patients have or will subsequently develop Bz resistance. Thus, as Bz resistance remains a significant challenge, research efforts are needed to identify novel biomarkers of early Bz resistance, particularly when an early therapeutic intervention can be initiated. Recent advances in MM research indicate that genomic data can be extracted to identify novel biomarkers that can be utilized to select more effective, personalized treatment protocols for individual patients. Computationally integrating large patient databases with data from whole transcriptome profiling and laboratory-based models can potentially revolutionize our understanding of MM disease mechanisms. This systems-wide approach can provide rational therapeutic targets and novel biomarkers of risk and treatment response. In this review, we discuss the use of high-content datasets (predominantly gene expression profiling) to identify novel biomarkers of treatment response and resistance to Bz in MM.

Bortezomib Resistance Can Be Reversed by Induced Expression of Plasma Cell Maturation Markers in a Mouse In Vitro Model of Multiple Myeloma

Multiple myeloma (MM), the second most common hematopoietic malignancy, remains an incurable plasma cell (PC) neoplasm. While the proteasome inhibitor, bortezomib (Bz) has increased patient survival, resistance represents a major treatment obstacle as most patients ultimately relapse becoming refractory to additional Bz therapy. Current tests fail to detect emerging resistance; by the time patients acquire resistance, their prognosis is often poor. To establish immunophenotypic signatures that predict Bz sensitivity, we utilized Bz-sensitive and -resistant cell lines derived from tumors of the Bcl-XL/Myc mouse model of PC malignancy. We identified significantly reduced expression of two markers (CD93, CD69) in “acquired” (Bz-selected) resistant cells. Using this phenotypic signature, we isolated a subpopulation of cells from a drug-naïve, Bz-sensitive culture that displayed “innate” resistance to Bz. Although these genes were identified as biomarkers, they may indicate a mechanism for Bz-resistance through the loss of PC maturation which may be induced and/or selected by Bz. Significantly, induction of PC maturation in both “acquired” and “innate” resistant cells restored Bz sensitivity suggesting a novel therapeutic approach for reversing Bz resistance in refractory MM.

Functional convergence of histone methyltransferases EHMT1 and KMT2C involved in intellectual disability and autism spectrum disorder

Kleefstra syndrome, caused by haploinsufficiency of euchromatin histone methyltransferase 1 (EHMT1), is characterized by intellectual disability (ID), autism spectrum disorder (ASD), characteristic facial dysmorphisms, and other variable clinical features. In addition to EHMT1 mutations, de novo variants were reported in four additional genes (MBD5, SMARCB1, NR1I3, and KMT2C), in single individuals with clinical characteristics overlapping Kleefstra syndrome. Here, we present a novel cohort of five patients with de novo loss of function mutations affecting the histone methyltransferase KMT2C. Our clinical data delineates the KMT2C phenotypic spectrum and reinforces the phenotypic overlap with Kleefstra syndrome and other related ID disorders. To elucidate the common molecular basis of the neuropathology associated with mutations in KMT2C and EHMT1, we characterized the role of the Drosophila KMT2C ortholog, trithorax related (trr), in the nervous system. Similar to the Drosophila EHMT1 ortholog, G9a, trr is required in the mushroom body for short term memory. Trr ChIP-seq identified 3371 binding sites, mainly in the promoter of genes involved in neuronal processes. Transcriptional profiling of pan-neuronal trr knockdown and G9a null mutant fly heads identified 613 and 1123 misregulated genes, respectively. These gene sets show a significant overlap and are associated with nearly identical gene ontology enrichments. The majority of the observed biological convergence is derived from predicted indirect target genes. However, trr and G9a also have common direct targets, including the Drosophila ortholog of Arc (Arc1), a key regulator of synaptic plasticity. Our data highlight the clinical and molecular convergence between the KMT2 and EHMT protein families, which may contribute to a molecular network underlying a larger group of ID/ASD-related disorders.

Bosutinib, a Lyn/Btk inhibiting tyrosine kinase inhibitor, is ineffective in advanced systemic mastocytosis

vival (P 5 0.08). Our goal is to improve the nationwide survival of myeloma patients. We have established a national registry of all myeloma patients to identify areas of intervention. A population-based survey showed no difference in causes of death among patients ineligible for HDT, who died within 30 days from diagnosis as compared to patients who died between 31 and 180 days. Patients who died within 30 days had higher LDH levels and lower levels of plasma albumin as compared to those patients who died within 31– 180 days, and may reflect patients with high tumor burden and a high degree of stress caused by severe infection, impaired kidney, and liver function. When compared with patients who died later than 180 days of diagnosis, WHO performance status was worse in patients with early death. A clinician will not find these results surprising. However, our data may imply that elderly patients with multiple myeloma are diagnosed with delay and a nationwide focus is now on accelerated diagnosis to clarify whether individuals presenting with an M-protein in blood or urine or with myeloma associated symptoms are suffering from myeloma. Furthermore, our survey showed that among patients with early deaths the most common causes of deaths were infections, cardiovascular failure, and renal failure. Standard induction treatment for patients ineligible for HDT now includes adjustment of chemotherapy dose according to co-morbidity and a proteasome inhibitor. Clinicians use several approaches to avoid life-threatening infections among the elderly such as granulocyte colony-stimulating factor (G-CSF) in patients with neutropenia and supportive treatment with immunoglobulins. To provide the patients with the best antibiotic treatment in future we are now conducting a nationwide prospective study on prophylactic antibiotic treatment.

Phenotypic and functional characterization of a bortezomib-resistant multiple myeloma cell line by flow and mass cytometry.

Abstract Multiple myeloma (MM) is an incurable malignant plasma cell neoplasm. Proteasome inhibitors including Bortezomib (Bz) are used to treat MM, and treatment failure due to drug resistance occurs. Bz-sensitive and -resistant MM cells have distinct immunophenotypic signatures that correlate with clinical outcome. These changes can be identified by fluorescence-based cytometry (FBC), however, FBC is rarely used in predicting Bz resistance. Mass cytometry (MC) is a recently developed variation of flow cytometry that detects heavy metal-ion tagged antibodies using time-of-flight mass spectrometry allowing for detection of up to 38 epitopes simultaneously in a single cell, without significant overlap, exceeding the dimensionality of FBC 3–4-fold. Here, we compared FBC and MC in the immunophenotypic characterization of Bz-sensitive and -resistant human MM cell line U266. We show that Bz-resistant cells are associated with the loss of CD56 and CD66a adhesion molecules as well as an activation signature.

CNV-RF Is a Random Forest–Based Copy Number Variation Detection Method Using Next-Generation Sequencing

Simultaneous detection of small copy number variations (CNVs) (<0.5 kb) and single-nucleotide variants in clinically significant genes is of great interest for clinical laboratories. The analytical variability in next-generation sequencing (NGS) and artifacts in coverage data because of issues with mappability along with lack of robust bioinformatics tools for CNV detection have limited the utility of targeted NGS data to identify CNVs. We describe the development and implementation of a bioinformatics algorithm, copy number variation-random forest (CNV-RF), that incorporates a machine learning component to identify CNVs from targeted NGS data. Using CNV-RF, we identified 12 of 13 deletions in samples with known CNVs, two cases with duplications, and identified novel deletions in 22 additional cases. Furthermore, no CNVs were identified among 60 genes in 14 cases with normal copy number and no CNVs were identified in another 104 patients with clinical suspicion of CNVs. All positive deletions and duplications were confirmed using a quantitative PCR method. CNV-RF also detected heterozygous deletions and duplications with a specificity of 50% across 4813 genes. The ability of CNV-RF to detect clinically relevant CNVs with a high degree of sensitivity along with confirmation using a low-cost quantitative PCR method provides a framework for providing comprehensive NGS-based CNV/single-nucleotide variant detection in a clinical molecular diagnostics laboratory.