Why Proteasome Inhibitors Cannot ERADicate Multiple Myeloma

Cancer Cell

Previews Why Proteasome Inhibitors Cannot ERADicate Multiple Myeloma Robert Z. Orlowski1,2,* 1Department

of Lymphoma/Myeloma of Experimental Therapeutics The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.ccr.2013.08.014 2Department

Proteasome inhibitors are key parts of our armamentarium against multiple myeloma, but the disease can become resistant through poorly defined mechanisms. In this issue of Cancer Cell, Leung-Hagesteijn and colleagues describe XBP1s subpopulations of tumor cells that are resistant to bortezomib and may account for therapeutic failures in the clinic. Multiple myeloma is a clonal proliferation of neoplastic plasma cells that can present clinically with hypercalcemia, renal insufficiency, anemia, bony lesions, bacterial infections, hyperviscosity, and amyloidosis. Worldwide, approximately 86,000 patients will be diagnosed each year with myeloma, which, in many areas, makes it the second most common hematologic malignancy, while about 63,000 patients die every year from diseaserelated complications (Becker, 2011). In the United States, due to an aging populace, it is anticipated that the number of cases of myeloma will grow by 57% between 2010 and 2030 (Smith et al., 2009), ranking myeloma behind only stomach and liver cancer in the rate of growth of new cases. These facts indicate that multiple myeloma is, and will continue to be, a significant source of morbidity and mortality. Fortunately, a number of advances over the past decade have dramatically improved patient outcomes. Among these are the advent of novel chemotherapeutics, including the immunomodulatory agents thalidomide, lenalidomide, and pomalidomide, and the proteasome inhibitors (Moreau et al., 2012) bortezomib and carfilzomib. All of these drugs have garnered regulatory approvals and are used in patients with newly diagnosed, relapsed, or relapsed/refractory disease and have contributed to a doubling of the median overall survival in many populations. This has been fueled by a greater understanding of the pathobiology of myeloma and by the development of more physiologically relevant preclinical models, allowing for greater success in translating laboratory concepts into

patient-focused therapies. Indeed, a number of the most potent combination regimens in use have been rationally developed based on studies of the mechanisms of action of these drug classes (Mahindra et al., 2012). Despite these significant advances, patients are currently treated on an empiric basis, typically with the regimens that have the highest response rates to which they have access, rather than based on the principles of precision medicine (Mendelsohn, 2013). The proteasome inhibitors bortezomib and carfilzomib as single agents in the relapsed and refractory settings both have response rates of approximately 25%, indicating a role for innate or primary resistance in modulating their activity. Response rates and, even more importantly, response durability can be improved by using them earlier in the disease process and incorporating them into combination regimens. However, no clinically relevant biomarkers have been validated that would allow practitioners to prospectively identify patients with disease that is most likely to respond to proteasome inhibition, and the same is true for the immunomodulatory drugs. In addition, even in patients whose disease initially responds, resistance to proteasome inhibitor-based therapy emerges in many, indicating a role for acquired or secondary resistance through incompletely understood mechanisms. As a result, some patients are exposed to the toxicities of these agents without a benefit, delaying their access to more effective options, and wasting precious healthcare resources. Clinically, these factors contribute to the natural history

of the disease, which follows a course characterized by multiple relapses and decreasing durations of benefit with each subsequent line of therapy. The work of Leung-Hagesteijn et al. (2013) in this issue of Cancer Cell may, however, begin to change the current status quo. Early attempts to identify mechanisms of innate bortezomib resistance implicated a role for overexpression of proteasome genes and especially of PSMD4 (Shaughnessy et al., 2011). Later, studies of acquired resistance models suggested mutations of the b5 proteasome subunit targeted by bortezomib were involved. However, these b5 mutations were subsequently not detected in patient samples, as indicated in the introduction by Leung-Hagesteijn et al. (2013). More recently, induction of signaling through the insulin-like growth factor (IGF)/IGF-1 receptor pathway has been implicated in acquired resistance (Kuhn et al., 2012), and clinical trials to test this possibility are planned. Finally, upregulation of a cluster of NRF2 oxidative stress response genes, including CHOP (DDIT3), was identified as a possible resistance signature in studies of murine myeloma models and clinically annotated gene expression profiling data sets (Stessman et al., 2013). In contrast, Leung-Hagesteijn et al. (2013) provide a different perspective starting from kinome-wide RNA interference studies intended to identify modulators of proteasome inhibitor sensitivity. They report that suppression of inositolrequiring enzyme 1 (IRE1) and its downstream effector, X-box binding protein 1 (XBP1), was associated with bortezomib

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Cancer Cell

Previews Myeloma Tumor Cell Compartment Acvated B-cell

CD 30+ XBP1s-

PAX5 XBP1s-

Pre-plasmablast

PAX5lo

Plasmablast

Plasma cell

CD 38+ XBP1s++

XBP1s+

XBP1s-

CD138lo

CD 20+

CD27

CD138+

lo C CD27

IL6R

CD 20lo

ERADlo cells

Y

B-cell

ERADmod cells

ERADhi cells

Bortezomib-based chemotherapy

XBP1s-

XBP1s-

XBP1sApoptosis

Bortezomib-resistant relapse Figure 1. Model of Proteasome Inhibitor Resistance Patients with multiple myeloma may have a number of neoplastic subpopulations, including B cells, activated B cells, pre-plasmablasts, plasmablasts, and plasma cells. B cells are typically cluster differentiation antigen 20+ (CD20+)/CD27+/paired box 5+ (PAX5+) and do not have spliced X-box binding protein 1 message (XBP-1 s ), whereas activated B cells are similar but express lower levels of CD20 and can be CD30+. Neither B cells nor activated B cells are involved in substantial immunoglobulin synthesis, and both therefore have relatively low levels of proteasome-assisted, endoplasmic reticulum-associated protein degradation (ERADlo). Pre-plasmablasts are probably CD27lo/PAX5lo/CD30+/XBP-1 s , express the interleukin 6 receptor (IL-6R+), produce low levels of monoclonal protein (Y), and therefore have modest levels of ERAD activity (ERADmod). Plasmablasts are CD38+/CD138lo/IL-6R+/XBP-1 s+, secrete increased levels of monoclonal protein, and therefore have higher ERAD activity (ERADhi). Finally, terminally differentiated plasma cells are CD38+/CD138+/IL-6R+/XBP-1 s+, produce high levels of monoclonal protein, and have the highest levels of ERAD activity. These phenotypic characteristics are taken from the work of Leung-Hagesteijn et al. (2013) and from other literature sources. Plasmablasts and plasma cells are sensitive to proteasome inhibitors such as bortezomib because of their high level of immunoglobulin synthesis, leading to high levels of ERAD-related stress, which triggers dependence on the unfolded protein response (UPR). B cells, activated B cells, and pre-plasmablasts, in part because they are XBP-1 s , produce less immunoglobulin and are therefore less susceptible. Treatment with bortezomib eradicates plasmablasts and plasma cells, which initially may make up the bulk of the tumor compartment and results in elimination of the monoclonal protein marker. However, B cells, activated B cells, and pre-plasmablasts, which initially are a minor component of the tumor compartment, survive. After expansion of these cells, patients may then develop clinical relapses that are bortezomib-resistant with disease that is hypo- or nonsecretory and may require a different treatment approach.

resistance. This, to some extent, challenges a current dogma, because IRE1 and XBP1 make up one arm of the unfolded protein response (UPR). Indeed, UPR activation was felt to represent an adaptation to proteasome-assisted endoplasmic reticulum-associated degradation (ERAD), which causes ER stress in plasma cells due to their large loads of misfolded proteins. These IRE1lo and XBP1lo cells showed lower expression of plasma cell maturation markers, lower immunoglobulin synthesis, and lower levels of UPR activation, consistent with a lower stress level and proteasome

load, which would make them resistant to proteasome inhibitors. Moreover, plasma cell maturation markers were enriched in samples from patients who responded to bortezomib-based therapy and reduced in those with no response to bortezomib. Finally, analysis of patient samples revealed the presence of up to five tumor cell subpopulations, including B cells, activated B cells, pre-plasmablasts, plasmablasts, and plasma cells, which would be expected to have different sensitivities to bortezomib (Figure 1). Consistent with this possibility, myeloma B cell and pre-plasma-

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blast progenitors were found to survive proteasome inhibition and be enriched in samples that were refractory to bortezomib. These findings have a number of key implications for innate and acquired proteasome inhibitor resistance. First, they suggest that the seeds for bortezomib resistance, and cross-resistance with other proteasome inhibitors, are already present in perhaps all patients and that this phenotype will inevitably emerge through the attrition of bortezomib-sensitive plasmablasts and plasma cells (Figure 1). Second, they support the possibility that gene expression profiling or whole genome sequencing to identify the baseline proportion of these progenitor cells present in patients prior to initiation of therapy may predict the extent of benefit to be expected from bortezomib. Finally, they imply that potentially curative strategies will need to target both the committed plasma cells and the progenitor, or stem cell-like cells. While these hypotheses are being tested, the current findings will also need to be validated in a wider array of primary samples and additional gene expression profiling data sets. One question that needs to be answered is whether the same proteasome inhibitor-resistant progenitor cells survive therapy with bortezomib in combination with other agents. This would be especially of interest for the combination of bortezomib and lenalidomide, because the latter may target clonogenic side-population cells (Jakubikova et al., 2011), and this regimen is one of our current standards of care against myeloma. Another important question is whether it would be possible to induce differentiation of progenitor cells to more committed plasma cells, such as through epigenetic therapies, to enhance or restore proteasome inhibitor sensitivity. Finally, it is likely that multiple mechanisms contribute to the development of the proteasome inhibitor-resistant phenotype. For example, while the current studies indicate that cells with a decreased proteasome load are resistant, it is also possible that changes that enhance plasma cell proteasome capacity would antagonize the beneficial effects of bortezomib and carfilzomib. Taken together, however, these findings do provide an important direction for future preclinical and clinical

Cancer Cell

Previews studies that will hopefully bring us to the point that we can apply precision medicine to the therapy of multiple myeloma. ACKNOWLEDGMENTS R.Z.O. would like to acknowledge support from the National Cancer Institute in the form of The MD Anderson Cancer Center SPORE in Multiple Myeloma (P50 CA142509) and the Southwest Oncology Group (U10 CA032102). R.Z.O. has received research funding from Bristol-Myers Squibb, Celgene Corporation, Millennium: The Takeda Oncology Company, and Onyx Pharmaceuticals. Also, R.Z.O. has served on advisory boards for Array Biopharma, Bristol-Myers Squibb, Celgene Corporation, Genentech, Merck, Millennium: The Takeda Oncology Company, and Onyx Pharmaceuticals.

Mendelsohn, J. (2013). J. Clin. Oncol. 31, 1904– 1911.

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RASAL2: Wrestling in the Combat of Ras Activation Jia Shen,1,2 Yan Wang,1 and Mien-Chie Hung1,2,3,* 1Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA 2The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030, USA 3Center for Molecular Medicine and Graduate Institute of Cancer Biology, China Medical University, Taichung 402, Taiwan *Correspondence: [email protected] http://dx.doi.org/10.1016/j.ccr.2013.08.024

Oncogenic activation of Ras proteins due to missense mutations is frequently detected in human cancers but rarely in breast cancer. In this issue of Cancer Cell, McLaughlin and colleagues report that ablation of the GasGAP gene, RASAL2, is an alternative mechanism by which Ras becomes activated in breast cancer. Ras GTPases are essential components of signaling pathways that emanate cues from cell surface receptors to regulate diverse cellular processes, including cell cycle progression, cell survival, actin cytoskeletal organization, cell polarity and movement, as well as vesicular and nuclear transport (Vigil et al., 2010). Ras proteins (H-Ras, N-Ras, and K-Ras), together with their two key regulators (guanine nucleotide exchange factors/ GEFs and GTPase-activating proteins/ GAPs), constitute cellular binary switches that cycle between ‘‘on’’ and ‘‘off’’ conformations conferred by the loading of GTP or GDP, respectively. The transition between the active GTP-bound and inactive GDP-bound states of Ras GTPases is controlled by GEFs, which promote the activation of Ras proteins by stimulating

GDP for GTP exchange, and GAPs, which terminate the activation status by accelerating Ras-mediated GTP hydrolysis (Bos et al., 2007; Tcherkezian and LamarcheVane, 2007). Ras GTPases and cancer are tightly associated, as high frequency of mutational activation of Ras proteins is observed in 33% of human cancers. The intrinsic GTP hydrolysis activity of Ras is the predominant target of the most common somatic mutations that are found in the oncogenic variants of RAS alleles (Pylayeva-Gupta et al., 2011). Specifically, oncogenic substitution in residue G12 or G13 results in pronounced attenuation of intrinsic GTP hydrolysis, which leads to the persistence of the GTP-bound state of Ras and subsequently activates a multitude of Ras-dependent downstream effector

pathways. Beyond Ras, hyperactivation of GEFs and functional deregulation, including suppression and loss-of-function mutations of GAPs, have also been suggested to play important roles in cancer progression (Vigil et al., 2010). Despite the prevalence of oncogenic RAS mutations in human cancers, KRAS, H-RAS, and N-RAS are rarely mutated in breast cancer (Karnoub and Weinberg, 2008). Nonetheless, the Ras/ ERK pathway is hyperactivated in more than half of breast cancers and has been implicated in tumor progression and recurrence (von Lintig et al., 2000), suggesting that Ras proteins may be more frequently activated by alternative mechanisms in this type of tumors. A new study by McLaughlin et al. (2013) in this issue of Cancer Cell uncovers the role of RASAL2,

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