Targeting the insulin-like growth factor-1 receptor to overcome bortezomib resistance in preclinical models of multiple myeloma.

Proteasome inhibition with bortezomib is a validated approach to the treatment of multiple myeloma, but drug resistance often emerges and limits its utility in the retreatment setting. To begin to identify some of the mechanisms involved, we developed bortezomib-resistant myeloma cell lines that, unlike previously reported models, showed no ß5 subunit mutations. Instead, up-regulation of the insulin-like growth factor (IGF)-1 axis was identified, with increased autocrine and paracrine secretion of IGF-1, leading to increased activation of the IGF-1 receptor (IGF-1R). Exogenous IGF-1 reduced cellular sensitivity to bortezomib, whereas pharmacologic or small hairpin RNA-mediated IGF-1R suppression enhanced bortezomib sensitivity in cell lines and patient samples. In vitro studies with OSI-906, a clinically relevant dual IGF-1R and insulin receptor inhibitor, showed it acted synergistically with bortezomib, and potently resensitized bortezomib-resistant cell lines and patient samples to bortezomib. Importantly, OSI-906 in combination with bortezomib also overcame bortezomib resistance in an in vivo model of myeloma. Taken together, these data support the hypothesis that signaling through the IGF-1/IGF-1R axis contributes to acquired bortezomib resistance, and provide a rationale for combining bortezomib with IGF-1R inhibitors like OSI-906 to overcome or possibly prevent the emergence of bortezomib-refractory disease in the clinic.

Evidence of a role for activation of Wnt/beta-catenin signaling in the resistance of plasma cells to lenalidomide.

Lenalidomide plays an important role in our chemotherapeutic armamentarium against multiple myeloma, in part by exerting direct anti-proliferative and pro-apoptotic effects. Unfortunately, long-term exposure leads to the development of drug resistance through unknown mechanisms, and we therefore sought to identify pathways that could be responsible for this phenotype. Chronic drug exposure produced myeloma cell lines that were tolerant of the direct effects of lenalidomide, with a degree of resistance of up to 2,500-fold. Gene expression profiling and pathway analysis identified dysregulation of the Wnt/ß-catenin pathway as a consistent change across four independent cell isolates, and a pair of primary plasma cell samples. Acute drug treatment also increased ß-catenin transcription by 3-fold or more, and both acute and chronic exposure resulted in enhanced accumulation of ß-catenin protein by up to 20-fold or more. This produced Wnt/ß-catenin pathway activation, as judged by increased activity of a lymphoid enhancer factor/T-cell factor promoter reporter, and enhanced accumulation of the downstream targets cyclin D1 and c-Myc. Components of the ß-catenin destruction complex were also impacted by lenalidomide, which suppressed casein kinase 1α expression while augmenting glycogen synthase kinase 3α/ß phosphorylation. Stimulation of Wnt/ß-catenin signaling with recombinant Wnt-3a, or by overexpression of ß-catenin, reduced the anti-proliferative activity of lenalidomide. Conversely, suppression of ß-catenin with small hairpin RNAs restored plasma cell sensitivity to lenalidomide. Together, these findings support the hypothesis that lenalidomide mediates activation of Wnt/ß-catenin signaling in plasma cells as a mechanism of inducible chemoresistance through effects at the transcriptional and post-translational levels.

Blockade of interleukin-6 signalling with siltuximab enhances melphalan cytotoxicity in preclinical models of multiple myeloma.

Signalling through the interleukin (IL)-6 pathway induces proliferation and drug resistance of multiple myeloma cells. We therefore sought to determine whether the IL-6-neutralizing monoclonal antibody siltuximab, formerly CNTO 328, could enhance the activity of melphalan, and to examine some of the mechanisms underlying this interaction. Siltuximab increased the cytotoxicity of melphalan in KAS-6/1, INA-6, ANBL-6, and RPMI 8226 human myeloma cell lines (HMCLs) in an additive-to-synergistic manner, and sensitized resistant RPMI 8226.LR5 cells to melphalan. These anti-proliferative effects were accompanied by enhanced activation of drug-specific apoptosis in HMCLs grown in suspension, and in HMCLs co-cultured with a human-derived stromal cell line. Siltuximab with melphalan enhanced activation of caspase-8, caspase-9, and the downstream effector caspase-3 compared with either of the single agents. This increased induction of cell death occurred in association with enhanced Bak activation. Neutralization of IL-6 also suppressed signalling through the phosphoinositide 3-kinase/Akt pathway, as evidenced by decreased phosphorylation of Akt, p70 S6 kinase and 4E-BP1. Importantly, the siltuximab/melphalan regimen demonstrated enhanced anti-proliferative effects against primary plasma cells derived from patients with myeloma, monoclonal gammopathy of undetermined significance, and amyloidosis. These studies provide a rationale for translation of siltuximab into the clinic in combination with melphalan-based therapies.

Targeted inhibition of the immunoproteasome is a potent strategy against models of multiple myeloma that overcomes resistance to conventional drugs and nonspecific proteasome inhibitors.

Proteasome inhibition is a validated strategy for therapy of multiple myeloma, but this disease remains challenging as relapses are common, and often associated with increasing chemoresistance. Moreover, nonspecific proteasome inhibitors such as bortezomib can induce peripheral neuropathy and other toxicities that may compromise the ability to deliver therapy at full doses, thereby decreasing efficacy. One novel approach may be to target the immunoproteasome, a proteasomal variant found predominantly in cells of hematopoietic origin that differs from the constitutive proteasome found in most other cell types. Using purified preparations of constitutive and immunoproteasomes, we screened a rationally designed series of peptidyl-aldehydes and identified several with relative specificity for the immunoproteasome. The most potent immunoproteasome-specific inhibitor, IPSI-001, preferentially targeted the beta1(i) subunit of the immunoproteasome in vitro and in cellulo in a dose-dependent manner. This agent induced accumulation of ubiquitin-protein conjugates, proapoptotic proteins, and activated caspase-mediated apoptosis. IPSI-001 potently inhibited proliferation in myeloma patient samples and other hematologic malignancies. Importantly, IPSI-001 was able to overcome conventional and novel drug resistance, including resistance to bortezomib. These findings provide a rationale for the translation of IPSIs to the clinic, where they may provide antimyeloma activity with greater specificity and less toxicity than current inhibitors.

Targeting the p27 E3 ligase SCF(Skp2) results in p27- and Skp2-mediated cell-cycle arrest and activation of autophagy.

Decreased p27(Kip1) levels are a poor prognostic factor in many malignancies, and can occur through up-regulation of SCF(Skp2) E3 ligase function, resulting in enhanced p27 ubiquitination and proteasome-mediated degradation. While proteasome inhibitors stabilize p27(Kip1), agents inhibiting SCF(Skp2) may represent more directly targeted drugs with the promise of enhanced efficacy and reduced toxicity. Using high-throughput screening, we identified Compound A (CpdA), which interfered with SCF(Skp2) ligase function in vitro, and induced specific accumulation of p21 and other SCF(Skp2) substrates in cells without activating a heat-shock protein response. CpdA prevented incorporation of Skp2 into the SCF(Skp2) ligase, and induced G(1)/S cell-cycle arrest as well as SCF(Skp2)- and p27-dependent cell killing. This programmed cell death was caspase-independent, and instead occurred through activation of autophagy. In models of multiple myeloma, CpdA overcame resistance to dexamethasone, doxorubicin, and melphalan, as well as to bortezomib, and also acted synergistically with this proteasome inhibitor. Importantly, CpdA was active against patient-derived plasma cells and both myeloid and lymphoblastoid leukemia blasts, and showed preferential activity against neoplastic cells while relatively sparing other marrow components. These findings provide a rational framework for further development of SCF(Skp2) inhibitors as a novel class of antitumor agents.

Targeted inhibition of interleukin-6 with CNTO 328 sensitizes pre-clinical models of multiple myeloma to dexamethasone-mediated cell death.

Interleukin (IL)-6-mediated signalling attenuates the anti-myeloma activity of glucocorticoids (GCs). We therefore sought to evaluate whether CNTO 328, an anti-IL-6 monoclonal antibody in clinical development, could enhance the apoptotic activity of dexamethasone (dex) in pre-clinical models of myeloma. CNTO 328 potently increased the cytotoxicity of dex in IL-6-dependent and -independent human myeloma cell lines (HMCLs), including a bortezomib-resistant HMCL. Isobologram analysis revealed that the CNTO 328/dex combination was highly synergistic. Addition of bortezomib to CNTO 328/dex further enhanced the cytotoxicity of the combination. Experiments with pharmacologic inhibitors revealed a role for the p44/42 mitogen-activated protein kinase pathway in IL-6-mediated GC resistance. Although CNTO 328 alone induced minimal cell death, it potentiated dex-mediated apoptosis, as evidenced by increased activation of caspases-8, -9 and -3, Annexin-V staining and DNA fragmentation. The ability of CNTO 328 to sensitize HMCLs to dex-mediated apoptosis was preserved in the presence of human bone marrow stromal cells. Importantly, the increased activity of the combination was also seen in plasma cells from patients with GC-resistant myeloma. Taken together, our data provide a strong rationale for the clinical development of the CNTO 328/dex regimen for patients with myeloma.

Proteasome inhibitors in cancer therapy: lessons from the first decade.

The ubiquitin-proteasome pathway is involved in intracellular protein turnover, and its function is crucial to cellular homeostasis. First synthesized as probes of proteolytic processes, proteasome inhibitors began to be thought of as potential drug candidates when they were found to induce programmed cell death preferentially in transformed cells. They made their first leap into the clinic to be tested as therapeutic agents 10 years ago, and since then, great strides have been made in defining their mechanisms of action, their clinical efficacy and toxicity, and some of their limitations in the form of resistance pathways. Validation of the ubiquitin-proteasome pathway as a target for cancer therapy has come in the form of approvals of the first such inhibitor, bortezomib, for relapsed/refractory multiple myeloma and mantle cell lymphoma, for which this agent has become a standard of care. Lessons learned from this first-in-class agent are now being applied to the development of a new generation of proteasome inhibitors that hold the promise of efficacy in bortezomib-resistant disease and possibly in a broader spectrum of diseases. This saga provides a salient example of the promise of translational medicine and a paradigm by which other agents may be successfully brought from the bench to the bedside.

Inhibition of interleukin-6 signaling with CNTO 328 enhances the activity of bortezomib in preclinical models of multiple myeloma.

PURPOSE: Inhibition of the proteasome leads to the activation of survival pathways in addition to those that promote cell death. We hypothesized that down-regulation of interleukin-6 (IL-6) signaling using the monoclonal antibody CNTO 328 would enhance the antitumor activity of the proteasome inhibitor bortezomib in multiple myeloma by attenuating inducible chemoresistance. EXPERIMENTAL DESIGN: The cytotoxicity of bortezomib, CNTO 328, and the combination, along with the associated molecular changes, was assessed in IL-6-dependent and IL-6-independent multiple myeloma cell lines, both in suspension and in the presence of bone marrow stromal cells and in patient-derived myeloma samples. RESULTS: Treatment of IL-6-dependent and IL-6-independent multiple myeloma cell lines with CNTO 328 enhanced the cytotoxicity of bortezomib in a sequence-dependent fashion. This effect was additive to synergistic and was preserved in the presence of bone marrow stromal cells and in CD138(+) myeloma samples derived from patients with relative clinical resistance to bortezomib. CNTO 328 potentiated bortezomib-mediated activation of caspase-8 and caspase-9 and the common downstream effector caspase-3; attenuated bortezomib-mediated induction of antiapoptotic heat shock protein-70, which correlated with down-regulation of phosphorylated signal transducer and activator of transcription-1; and inhibited bortezomib-mediated accumulation of myeloid cell leukemia-1, an effect that was associated with down-regulation of phosphorylated signal transducer and activator of transcription-3. CONCLUSIONS: Taken together, our results provide a strong preclinical rationale for the clinical development of the bortezomib/CNTO 328 combination for patients with myeloma.

Anti-angiogenic and anti-tumor properties of proteasome inhibitors.

Tumor growth and metastasis depend on the formation of blood vessels, angiogenesis, to supply the developing mass with nutrients, oxygen, and waste removal. The proteasome, a massive multisubunit catabolic body, exerts a regulatory influence on angiogenesis. Inhibition of the proteasome activity has been found to inhibit angiogenesis and induce apoptosis in human cancer cells with limited toxicity to normal cells. Therefore, the dual action of angiogenesis inhibition and cell death induction makes proteasome inhibition an attractive modality for chemotherapy. A variety of proteasome inhibitors have been studied including: antibiotics such as lactacystin, the green tea polyphenols, and the boronic acid Velcade (MLN-341). Most recently, certain classes of copper compounds have been found to act as potent proteasome inhibitors. The potential of particular organic compounds, such as 8-hydroxyquinoline, to spontaneously bind with tumor cellular copper and form proteasome inhibitors provides a new modality of anti-proteasome and anti-angiogenesis chemotherapy. This review examines angiogenesis, the proteasome, representative proteasome inhibitors, and the emerging role of copper. The formation of new blood vessels, or angiogenesis, is an important and necessary function in both embryonic development and wound repair. Therefore, the ability to regenerate or form new vessels for blood flow is essential. The control of angiogenic pathways is tightly regulated in normal differentiated adult cells, which generally do not stimulate blood vessel growth unless injury occurs. However, cancerous tissues stimulate angiogenesis that in turn leads to increased tumor formation and possible metastases. Many of the factors involved in angiogenesis are regulated by the proteasome, which recently has become a focus in anti-cancer therapies due to its involvement in cell cycle and apoptosis control. Here we discuss angiogenesis and its relation to the proteasome. Additionally, current modalities of anti-angiogenic treatment, mainly proteasome inhibitory strategies, are reviewed. Furthermore, proteasome inhibitors, both natural and synthetic, and their anti-angiogenic effects as well as future approaches to anti-angiogenic chemotherapies are also discussed.

Direct inhibition of the ubiquitin-proteasome pathway by ester bond-containing green tea polyphenols is associated with increased expression of sterol regulatory element-binding protein 2 and LDL receptor.

Green tea has been shown to lower plasma cholesterol, associated with up-regulation of the low-density lipoprotein receptor (LDLR) although the responsible molecular mechanism is unknown. Previously, we reported that ester bond-containing green tea polyphenols (GTPs), such as (-)-epigallocatechin-3-gallate [(-)-EGCG], potently inhibit the tumor cellular proteasome activity, which may contribute to the cancer-preventative effect of green tea. In the current study, we hypothesize that the proteasome is a heart disease-associated molecular target of GTPs. We have shown that ester bond-containing GTPs, including (-)-EGCG, potently inhibit the proteasomal activity in intact hepatocellular carcinoma HepG2 and cervical carcinoma HeLa cells, as evident by accumulation of ubiquitinated proteins and three natural proteasome targets (p27, IkappaB-alpha and Bax). (-)-EGCG selectively inhibits the chymotrypsin-like, but not trypsin-like, activity of the proteasome. Associated with proteasome inhibition by ester bond-containing GTPs, there was a significant, time- and concentration-dependent increase in levels of the cleaved, activated, but not the precursor, form of sterol regulatory element-binding protein 2 (SREBP-2), an essential factor for LDLR transcription. Subsequently, LDL receptor expression was increased dramatically in HepG2 and HeLa cells treated with (-)-EGCG. Our results suggest that ester bond-containing GTPs inhibit ubiquitin/proteasome-mediated degradation of the active SREBP-2, resulting in up-regulation of LDLR. This identified molecular mechanism may be related to the previously reported cholesterol-lowering and heart disease-preventative effects of green tea.

The role of interleukin-2 receptor alpha in cancer.

Interleukin-2 (IL-2) is the major growth factor for activated T-lymphocytes and stimulates clonal expansion and maturation of these lymphocytes. IL-2 binds to its receptor complex, IL-2Ralpha, beta, and gamma chains, and exerts its effect via second messengers, mainly tyrosine kinases, which ultimately stimulate gene expression. The heterotrimerization of the receptor chains leads to high affinity binding for IL-2. The functional importance of IL-2Ralpha in hematopoietic cell systems is well known. However, the potential role that IL-2Ralpha plays in tumorigenesis is still not fully elucidated. Il-2Ralpha expression has been found in many types of cancers, including leukemia, lymphoma, lung, breast, head-and-neck and prostate. Recent evidence shows that high expression of IL-2Ralpha in tumors correlates with a poor prognosis for the patient. This review details the current known functions of IL-2Ralpha in cancer cells and some of the therapies used to combat IL-2Ralpha-mediated cell signaling.

Structure-activity relationships OF N-methylthiolated beta-lactam antibiotics with C3 substitutions and their selective induction of apoptosis in human cancer cells.

The development of novel anti-cancer drugs that induce apoptosis has long been a focus of drug discovery. Beta-lactam antibiotics have been used for over 60 years to fight bacterial infectious diseases with little or no side effects observed. Recently a new class of N-methylthiolated beta-lactams has been discovered that have potent activity against methicillin resistant Staphylococcus aureas. Most recently, we determined the potential effects of these N-thiolated beta-lactams on tumorigenic cell growth and found that they are apoptosis-inducers in human cancer cell lines. In the current study, we further determined the effects of the substitution of the O-methyl moiety on C3 and stereochemistry of the beta-lactams on the anti-proliferative and apoptosis-inducing abilities. We have found that lactam 18, in which C3 is substituted with an acrylate ester group, is a very effective proliferation inhibitor against human premalignant and malignant breast, leukemic, and simian virus 40-transformed fibroblast cells. Generally speaking, increasing the size of the moiety on C3 decreases its anti-proliferation potency, possibly indicating steric hindrance with the cellular target or decreased permeability through the cell membrane. We also found that the stereochemistry of the beta-lactams plays an important role in their potency. The 3S,4R isomers are more effective than their enantiomers (3R,4S), suggesting that 3S,4R configuration is more favorable for target interaction.

Dietary flavonoids as proteasome inhibitors and apoptosis inducers in human leukemia cells.

It has been shown that proteasome activity is required for cancer cell survival and consumption of fruits and vegetables is associated with decreased cancer risk. Previously, we reported that grape extract could inhibit proteasome activity and induce apoptosis in tumor cells. In this study, we examined the flavonoids apigenin, quercetin, kaempferol and myricetin for their proteasome-inhibitory and apoptosis-inducing abilities in human tumor cells. We report that apigenin and quercetin are much more potent than kaempferol and myricetin at: (i) inhibiting chymotrypsin-like activity of purified 20S proteasome and of 26S proteasome in intact leukemia Jurkat T cells; (ii) accumulating putative ubiquitinated forms of two proteasome target proteins, Bax and Inhibitor of nuclear factor kappabeta-alpha in Jurkat T cells and (iii) inducing activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase in Jurkat T cells. The proteasome-inhibitory abilities of these compounds correlated with their apoptosis-inducing potencies. Results from computational modeling of the potential interactions of these flavonoids to the chymotrypsin site (beta5 subunit) of the proteasome were consistent with the obtained proteasome-inhibitory activities. We found that the C(4) carbon may be a site of nucleophilic attack by the OH group of N-terminal threonine of proteasomal beta5 subunit and that the C(3) hydroxyl may alter the ability of these flavonoids to inhibit the proteasome. Finally, apigenin neither effectively inhibited the proteasome activity nor induced apoptosis in non-transformed human natural killer cells. Our results suggested that the proteasome may be a target of these dietary flavonoids in human tumor cells and that inhibition of the proteasome by flavonoids may be one of the mechanisms responsible for their cancer-preventive effects.

Evaluation of proteasome-inhibitory and apoptosis-inducing potencies of novel (-)-EGCG analogs and their prodrugs.

The anti-cancer and cancer-preventive effects of green tea and its main constituent (-)-epigallocatechin gallate [(-)-EGCG] are well documented by a variety of studies, including epidemiological, cell culture, animal, and clinical studies. While (-)-EGCG remains the most potent polyphenol in green tea, it is very unstable in neutral or alkaline conditions (i.e. physiologic pH). In an effort to discover more stable polyphenol proteasome inhibitors, we synthesized several novel (-)-EGCG analogs with -OH groups eliminated from the B- and/or D-rings. In addition, we also synthesized their putative prodrugs with -OH groups protected by peracetate that can be removed by cellular cytosolic esterases. We first examined the structure-activity relationship of these unprotected and protected compounds to their proteasome-inhibitory potentials. We found that decreasing -OH groups from either the B- or D-ring leads to diminished proteasome-inhibitory activity in vitro. However, in cultured tumor cells only the protected analogs were capable of potently inhibiting the proteasome activity. Furthermore, these protected analogs induced apoptotic cell death in a tumor cell-specific manner. The superior efficacy of the protected (-)-EGCG analogs indicates the formation of an entirely new compound(s) in intact tumor cells. These data suggest that the B-ring/D-ring peracetate-protected EGCG analogs have great potential to be developed into novel anti-cancer and cancer-preventive agents.

Green tea and tea polyphenols in cancer prevention.

The cancer-preventive effects of green tea and its main constituent (-)-epigallocatechin gallate [(-)-EGCG] are widely supported by results from epidemiological, cell culture, animal and clinical studies in the recent decade. In vitro cell culture studies show that tea polyphenols potently induce apoptotic cell death and cell cycle arrest in tumor cells but not in their normal cell counterparts. Green tea polyphenols affect several signal transduction pathways, including growth factor-mediated, the mitogen-activated protein kinase (MAPK)-dependent, and ubiquitin/proteasome degradation pathways. Epidemiological studies have suggested that the consumption of green tea lowers the risk of cancer. Various animal studies have revealed that treatment by green tea inhibits tumor incidence and multiplicity in different organ sites such as skin, lung, liver, stomach, mammary gland and colon. Phase I and II clinical trials were carried out recently to explore the anticancer effects of green tea in patients with cancer. At this time, more mechanistic research, animal studies, and clinical trials are necessary to further evaluate the role of green tea in cancer prevention.

Novel N-thiolated beta-lactam antibiotics selectively induce apoptosis in human tumor and transformed, but not normal or nontransformed, cells.

Historically, it has been shown that the beta-lactam antibiotics play an essential role in treating bacterial infections while demonstrating selectivity for prokaryotic cells. We recently reported that certain N-methylthio-substituted beta-lactam antibiotics had DNA-damaging and apoptosis-inducing activities in various tumor cells. However, whether these compounds affect human normal or nontransformed cells was unknown. In the current study, we first show that a lead compound (lactam 1) selectively induces apoptosis in human leukemic Jurkat T, but not in the nontransformed, immortalized human natural killer (NK) cells. Additionally, we screened a library of other N-methylthiolated beta-lactams to determine their structure-activity relationships (SARs), and found lactam 12 to have the highest apoptosis-inducing activity against human leukemic Jurkat T cells, associated with increased DNA-damaging potency. Furthermore, we demonstrate that lactam 12, as well as lactam 1, potently inhibits colony formation of human prostate cancer cells. We also show that lactam 12 induces apoptosis in human breast, prostate, and head-and-neck cancer cells. Finally, lactam 12 induces apoptosis selectively in Jurkat T and simian virus 40-transformed, but not in nontransformed NK and parental normal fibroblast, cells. Our results suggest that there is potential for developing this class of beta-lactams into novel anticancer agents.

A natural musaceas plant extract inhibits proteasome activity and induces apoptosis selectively in human tumor and transformed, but not normal and non-transformed, cells.

Animal studies have demonstrated that a dietary polyphenol known as tannic acid (TA) exhibits anticarcinogenic activity in chemically induced cancers. Most recently, we have reported that TA and ester-bond containing green tea polyphenols are potent proteasome inhibitors in vitro and in vivo. We hypothesize that CellQuest, a patented formula which contains high level of TA obtained from a musaceas (plantain) plant extract, will inhibit the tumor cell proteasome activity. Here, we report that a partially purified CellQuest fraction, S3, potently inhibits the proteasomal chymotrypsin-like activity of Jurkat T cell extracts in a concentration-dependent manner. Inhibition of the proteasome by S3 in leukemia Jurkat T, simian virus 40-transformed and prostate cancer LNCaP cells results in accumulation of ubiquitinated proteins and the natural proteasome substrate p27Kip1, followed by induction of apoptosis. In contrast, non-transformed, immortalized human natural killer cells and normal human fibroblasts are resistant to S3-mediated proteasome inhibition and apoptosis induction. Our present study suggests that CellQuest targets and inhibits the proteasome selectively in tumor cells, which may contribute to the claimed anticancer activity.

The immunoproteasome as a target in hematologic malignancies.

Suppression of proteasome function with the first-in-class small molecule inhibitor bortezomib is a rational therapeutic strategy against several hematologic malignancies, including multiple myeloma and mantle cell lymphoma. Second-generation inhibitors such as carfilzomib, ixazomib, and marizomib that, like bortezomib, target both the constitutive proteasome and the immunoproteasome, are also in clinical trials and showing encouraging activity. While the efficacy of these agents is well documented, toxicities associated with their use, such as peripheral neuropathy and gastrointestinal effects, can necessitate dose reductions or even discontinuations, possibly hampering their anti-neoplastic effects. These findings suggested that it could be possible to improve the therapeutic index of this class of drugs by specifically targeting only the immunoproteasome. Since the immunoproteasome is a unique target found in lymphoid-derived cells, immunoproteasome-specific inhibitors (IPSIs) could preserve efficacy while reducing treatment-emergent toxicities since they would spare other tissues with little to no immunoproteasome expression. This review discusses the current state of development of IPSIs, and the potential of using such agents for the treatment of hematologic malignancies.

Drug resistance to inhibitors of the human double minute-2 E3 ligase is mediated by point mutations of p53, but can be overcome with the p53 targeting agent RITA.

The human double minute (HDM)-2 E3 ubiquitin ligase plays a key role in p53 turnover and has been validated preclinically as a target in multiple myeloma (MM) and mantle cell lymphoma (MCL). HDM-2 inhibitors are entering clinical trials, and we therefore sought to understand potential mechanisms of resistance in lymphoid models. Wild-type p53 H929 MM and Granta-519 MCL cells resistant to MI-63 or Nutlin were generated by exposing them to increasing drug concentrations. MI-63-resistant H929 and Granta-519 cells were resistant to Nutlin, whereas Nutlin-resistant cells displayed cross-resistance to MI-63. These cells also showed cross-resistance to bortezomib, doxorubicin, cisplatin, and melphalan, but remained sensitive to the small molecule inhibitor RITA (reactivation of p53 and induction of tumor cell apoptosis). HDM-2 inhibitor-resistant cells harbored increased p53 levels, but neither genotoxic nor nongenotoxic approaches to activate p53 induced HDM-2 or p21. Resequencing revealed wild-type HDM-2, but mutations were found in the p53 DNA binding and dimerization domains. In resistant cells, RITA induced a G(2)-M arrest, upregulation of p53 targets HDM-2, PUMA, and NOXA, and PARP cleavage. Combination regimens with RITA and MI-63 resulted in enhanced cell death compared with RITA alone. These findings support the possibility that p53 mutation could be a primary mechanism of acquired resistance to HDM-2 inhibitors in MCL and MM. Furthermore, they suggest that simultaneous restoration of p53 function and HDM-2 inhibition is a rational strategy for clinical translation.