Protein kinase inhibitors against malignant lymphoma

1.1.1 SYK Inhibitors

The tyrosine kinase SYK is critical for normal B-cell development and proliferation and recent studies implicate this protein in the NHL pathogenesis. In particular, SYK is a key component of the BCR signaling pathway [8]. Given the vital role of SYK in BCR signaling, pharmacological or genetic targeting of SYK has been pursued as a therapeutic strategy for the treatment of NHL. SYK inhibition by using a chemical SYK inhibitor or by small interfering RNA (siRNA) results in potent inhibition of mammalian target of rapamycin (mTOR) activity in mantle cell lymphoma (MCL), large cell lymphoma and Burkitt’s lymphoma [9]. Targeting BCR signaling pathways appears to be a highly promising therapeutic strategy in B-cell lymphomas.

The majority of SYK inhibitors undergoing clinical development target the conserved ATP-binding site within the catalytic domain of the kinase [10–13]. Because of the similarities of the ATP pocket structures among different kinases, the ATP-competitive SYK inhibitors affect multiple TKs such as RET, KIT, JAK 1-3, FLT3, PDGFR-α, Aurora, LCK, KDR as well as adenosine A3 receptor and have off-target effects that lead to undesirable side effects such as hypertension, myelosuppression, teratogenicity, neutropenia or auto-immune diseases. Moreover, as SYK is widely distributed in different cell types, inhibiting its catalytic activity bears the risk of unwanted consequences on various physiological functions such as cell differentiation, adhesion and proliferation [11]. The lead SYK inhibitors in clinical trials include R406, R788, CG14979 and PRTO62607. Several of these ATP-binding site SYK inhibitors are under clinical trials in patients with B-cell lymphomas [10]. In addition, more specific small molecule substrate-binding (P) site SYK inhibitors as well as antisense oligonucleotides and siRNAs have also been developed to selectively knock down SYK [12]. The availability of both ATP-binding site inhibitors (e.g., R-406) and P-site inhibitors (e.g., C-61) of SYK may be particularly helpful in patients with SYK mutations that might influence inhibitor binding to ATP- and/or substrate-binding sites. Recent studies have established SYK kinase as a potential therapeutic target in NHL, CLL and acute myeloid leukemia.

Fostamatinib disodium (FosD/R788, (6-({5-fluoro-2-((3,4,5-trimethoxyphenyl)amino]pyrimidin-4-yl}amino)-2,2-dimethyl-3-oxo-2H-pyrido[3,2-b][1,4]oxazin-4(3H)-yl]methyl dihydrogen phosphate, disodium; AstraZeneca) is the first SYK inhibitor available that has been moved to the clinic. R788, a pro-drug of R406 is an oral ATP-competitive SYK inhibitor with demonstrated antitumor activity both in vitro and in vivo. In a Phase I/II clinical trial, Friedberg et al. [13] investigated R788 in patients with relapsed and refractory (R/R) B-cell NHL and CLL. In the Phase I part of the trial, two cohorts of six patients each received one of two dose levels, 200 or 250 mg, BID orally. All patients in cohort 1 had stable disease after treatment with R788 with a median duration of 5.3 months. In cohort 2, one patient with FL displayed a partial response (PR) with a response duration of 13.3 months. The dose-limiting toxicities were neutropenia, diarrhea and thrombocytopenia. In the Phase II study, 68 patients with recurrent B-NHL were treated with R788 (200 mg BID). The highest response rate (55%) was observed in patients with SLL/CLL. Objective response rates were also noted in DLBCL (22%), FL (10%) and MCL (11%) (Table 1). The median PFS was 4.2 months for all patients. Thus, differences in response rates among different NHL subtypes may be owing to biologic heterogeneity. Common toxicities observed in this study included diarrhea, fatigue and cytopenias. The authors speculated that inhibition of SYK may have disrupted the nodal microenvironment and led to increased trafficking of CLL cells out of nodal tissues and into the peripheral blood where they would then eventually die.

Because of the similarities of the ATP pocket structures among different kinases, the ATP-binding site SYK inhibitors affect multiple tyrosine kinases and have off-target activities. Indeed, hypertension, a common and potentially dangerous side effect of FosD, has been attributed to off-target inhibition of vascular endothelial growth factor receptor (VEGFR). Inhibitors targeting the substrate-binding sites of TKs are hoped to have enhanced specificity and potency [14]. Unlike available inhibitors of SYK targeting the ATP-binding site, C-61 targets the tyrosine kinase substrate-binding site of SYK [15] and thereby provides a unique opportunity to selectively target the SYK-dependent antiapoptotic blast cell survival machinery that is controlled by the TK activity of SYK, especially via tyrosine phosphorylation events leading to activation of signal transducer and activator of transcription 3 (STAT3) and PI3-kinase [16].

1.1.2 BTK Inhibitors

Like SYK, BTK is also intimately involved in multiple signal transduction pathways regulating survival, activation, proliferation and differentiation of B-lineage lymphoid cells [17]. A meta-analysis of cancer-associated gene expression changes utilizing the Oncomine database revealed a marked enrichment of the most discriminating BTK-dependent anti-apoptotic gene targets in 17 comparisons for diagnostic classes of human lymphomas and leukemias obtained from eight studies [18]. Consequently, BTK has emerged as a new molecular target for treatment of B-lineage NHL and leukemias.

Both covalent and noncovalent BTK inhibitors are being developed as therapeutic agents for various indications [18-20]. The clinically most advanced irreversible BTK inhibitor Ibrutinib/PCI-32765 ((R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one; Pharmacyclics) has demonstrated clinical activity against a variety of B-cell malignancies in ongoing Phase I/II trials including MCL, CLL, FL and DLBCL, with good tolerability. Likewise, dianilinopyrimidine-based irreversible BTK inhibitor AVL-292 (Avila Therapeutics/Celgene), a multikinase inhibitor, has promising in vitro activity against lymphoma cells. Dasatinib, a BCR-ABL kinase inhibitor that is US FDA approved for treatment of chronic myelogenous leukemia (CML), is a potent inhibitor of BTK [21]. Ibruti-nib/PCI-32765 is being investigated in patients with B-lineage lymphoid malignancies, including patients with relapsed/refractory CLL/small lymphocytic lymphoma (SLL), MCL, DLBCL, FL and multiple myeloma (MM). In Phase I/II studies, PCI-32765 showed encouraging clinical activity in patients with several types of B-cell lymphoma [22].

1.1.3 Anaplastic lymphoma kinase inhibitors

Anaplastic lymphoma kinase (ALK) is a RTK of the insulin receptor superfamily that transmits signals through a number of other important kinases, including PI3K and Janus kinase (JAK) [36].

Anaplastic large cell lymphoma (ALCL) is a rare type of aggressive T-cell lymphoma, comprising between 2.5 and 5% of all NHLs [37]. Approximately 50,000 cases of NHL are diagnosed annually in the US. Expression of ALK fusion proteins resulting from chromosomal translocations involving chromosome band 2p23 is a hallmark of ALCLS [38]. The most frequently detected ALCL translocation t(2;5) encodes a nucleophosmin (NPM)-ALK fusion protein with constitutive TK activity, which is clearly implicated in the pathogenesis of approximately 80% of cases of ALCL. ALK protein expression is an independent predictor of survival and serves as a useful biologic marker of a specific disease entity within the spectrum of ALCL [39]. ALK-positive ALCLs are highly responsive to cytotoxic drugs [40]. However, relapses occur and require salvage therapy.

The oncogenic role of the ALK fusion oncogene provides a potential avenue for therapeutic intervention. The development of ALK inhibitors, designed to interfere with ALK signaling, has revolutionized the treatment of lung cancer. Crizotinib (PF-02341066, Xalkori; 3-((R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl) pyridin-2-amine, Pfizer) is a multitargeted TKI, which was originally developed as an inhibitor of mesenchymal epithelial transition growth factor (c-MET); it is also a potent inhibitor of ALK phosphorylation and signal transduction [40]. Extremely promising Phase II results led to accelerated approval of crizotinib by the FDA for the treatment of patients with late-stage, nonsmall cell lung cancers (NSCLCs) who express the abnormal ALK gene. Crizotinib has progressed into several Phase III trials and a number of other ALK inhibitors are also in early stages of development.

Several second-generation ALK inhibitors are being studied in the clinic for patients with ALK-rearranged cancers. One of these, LDK378, is more potent and selective than Crizotinib and may overcome acquired resistance to Crizotinib. However, the response rate in ALK-positive NSCLC patients suggests that more potent ALK inhibitors like LDK378 may be useful in patients who relapse while on Crizotinib. ASP3026 (N2-[2-methoxy-4-[4-(4-methyl-1-piperazinyl)-1-piperidinyl]phenyl]-N4-[2-[(1-methylethyl)sulfonyl]phenyl]-1,3,5-triazine-2,4-diamine; Astellas Pharma) is being evaluated in Phase I trials in patients with advanced malignancies, B-cell lymphoma, solid tumors and ALK-positive tumors (NCT01284192). Development of a clinical grade anti-ALK antibody is also under way. Combining an anti-ALK antibody with ALK inhibitors might be more effective than either agent alone.

1.1.4 Inhibitors of the PI3K/AKT/mTOR pathway

The PI3K/AKT/mTOR pathway plays an important role in lymphomagenesis. This system regulates the expression of cyclin D1, c-Myc and STAT3 proteins, which are involved in the pathogenesis of B-cell NHL [41].

1.1.5 Src family kinase inhibitors

The Src family kinases (SFKs) are the largest family of nonreceptor TKs (NRTK) and one of the best-studied targets for cancer therapy [64,65].

Dasatinib (Sprycel, BMS-354825; N-(2chloro-6-methyl-phenyl)-2-(6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methylpyry-midin-4-ylamino)thiazole-5-carboxamide, Bristol-Myers Squibb) is an ATP-based competitor that was developed as a dual inhibitor of the Src family and Abl TKs (c-Src, Lck, Hck, Yes, Fgr, Lyn and Fyn) that is FDA approved for the treatment of CML. Approximately 95% of CML patients and 30% of acute lymphoblastic leukemia (ALL) patients are positive for the ‘Philadelphia chromosome,’ a mutation associated with tumors that are often difficult to treat with currently available therapies. Currently, five FDA-approved TKIs are available in the US–Gleevec (Imatinib), Tasigna (Nilotinib), Sprycel (Dasatinib), Bosulif (Bosutinib) and Ponatinib (Iclusig)–for people with CML or Philadelphia chromosome-positive ALL. Imatinib, Dasatinib, Nilotinib and Bosutinib all fail to be effective in patients with the T315I mutation. However, patients that express this mutation respond very well to Ponatinib.

Dasatinib is also a potent inhibitor of five other critical oncogenic TK families: SRC, c-KIT, PDGF receptors (α and β), BTK and ephrin (EPH) receptor kinases [66]. William et al. [67] reported the preliminary results of a Phase I/II trial on the safety and clinical activity of Dasatinib in NHL. The primary endpoint was MTD of Dasatinib in the Phase I stage and ORR in Phase II stage of the study. Dasatinib showed encouraging activity in heavily pretreated, recurrent or refractory NHL patients. Major toxicities noted were grade 3 pleural effusions and cytopenias. Dasatinib was found to be particularly effective in patients with peripheral T-cell lymphomas (PTCL); possibly because of high expression of PDGFR-α. After two cycles of treatment (150 mg PO 28-day cycles), the ORR was 32%, PFS was 17% at 1 year and 13% at 2 years. Overall survival was 60% at 1 year and 50% at 2 years. The two patients who sustained a CR had PTCL.

1.1.6 Protein Kinase C (PKC) inhibitors

Protein kinase C (PKC) is a family of serine/threonine kinases that regulate a variety of cell functions including proliferation, gene expression, cell cycle, differentiation, cytoskeletal organization, cell migration and apoptosis [68]. Small molecule approaches to inhibiting PKC have been directed toward the ATP-binding site or the diacylglycerol-binding site. Enzastaurin (LY317615, 3-(1-methylindol-3-yl)-4-[1-[1-(pyr-idin-2-ylmethyl)piperidin-4-yl]indol-3-yl]pyrrole-2,5-dione, Eli Lilly & Co.) is an oral serine/threonine inhibitor that suppresses cell signaling through the PKC-/PI3K/AKT pathways [69]. In a Phase II study, Morschhauser et al. [70] treated 60 patients with relapsed MCL with 500 mg of Enzastaurin daily. The most common side effect was fatigue. Although there were no documented antitumor responses, 37% remained progression free for > 3 months, and six patients were progression free for 6 months or more. In a Phase II study in R/R DLBCL, prolonged freedom from progression (FFP) was observed with little grade 3 toxicity. Preliminary results from a subsequent study in aggressive NHL also indicate single-agent activity. In a multicenter, Phase II study, Robertson et al. [71] treated 55 patients with relapsed or refractory DLBCL with Enzastaurin (500 mg daily) until disease progression or unacceptable toxicity. The primary endpoint was FFP for at least two cycles (2 months), and 22% of patients met this endpoint. The agent was well tolerated and the only grade 4 toxicity observed was hypomagnesemia. These encouraging data led to the initiation of Phase III trials with Enzastaurin in combination with Rituximab plus CHOP chemotherapy in patients with DLBCL (NCT00332202). In a Phase II study in stage III–IV FL patients receiving Enzastaurin 500 mg/d for up to 3 years or until progression, the ORR was 25% [72]. Clinical data with Enzastaurin in SLL/ CLL are quite limited with a single report on seven patients showing an ORR of 14.3% and a median PFS of 10.2 months [73]. The best results with Enzastaurin have been reported in WM patients [74]. In a Phase II study, 29 previously treated patients were given 500 mg/day Enzastaurin for 8 months or until disease progression. In all, 27.9% of the patients had a measurable response, with a decrease in monoclonal IgM > 25% in 37.9% of these cases.

1.1.7 Ras/Raf/MEK/ERK pathway inhibitors

The Ras/Raf/mitogen-activated kinase 1/2 (MEK1/2)/MAPK pathway is one of the most frequently dysregulated signaling cascades in cancer. Activating mutations of Ras and Raf occur frequently in both solid tumors and hematologic malignancies, leading to activation of their downstream targets MEK1/2 and extracellular signal-regulated kinase (ERK) 1/2. Ras mutation rates vary widely in hematopoietic cancers, with values ranging in leukemias from as low as 5% in CML to 27–70% in chronic myelomonocytic leukemia (CMML) [75]. A prevalence of activating mutations of both K-ras and N-ras has been described in MM [76]. The use of farnesyl transferase inhibitors (FTI) in the treatment of solid tumors has been disappointing despite promising preclinical studies. Unfortunately, the history of FTIs in the treatment of leukemias and lymphomas has also been disappointing [77]. Despite this, some clinical trials for hematopoietic malignancies are still ongoing that focus on the use of FTIs, either as single anti-Ras agents or in combination with different drugs targeting other components of relevant signaling pathways. The nonpepti-domimetic oral FTI, Tipifarnib (Zarnestra, R115777, 6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methylquinolin-2(1H)-one; Johnson & Johnson), has been used to treat patients with relapsed DLBCL, grade 3 FL or MCL. Of the 38 patients who were evaluated, 18% had PR and 21% had stable disease [78]. A second study in 11 MCL patients showed only one CR (9%) [79].

The multikinase inhibitor Sorafenib (BAY 43-9006, 4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino] phenoxy]-N-methyl-pyridine-2-carboxamide; Bayer/Onyx) was initially developed as a Raf-1 inhibitor but has subsequently been shown to inhibit multiple other kinases, including FLT3, PDGFR, VEGFR1 and VEGFR2 [80]. Ongoing clinical trials are testing the usefulness of anti-Ras monoclonal antibodies trial as well as various inhibitors of downstream kinases such as the Raf inhibitor Sorafenib or the MEK inhibitors AS703026 and GSK1120212.

1.1.8 JAK/STAT kinase inhibitors

The Janus kinase (JAK) family comprises cytoplasmic receptor-associated TKs that are involved in signal transduction pathways mediated by many cytokines and cytokine-like hormones [81].

Pacritinib (SB1518, 11-(2-pyrrolidin-1-yl-ethoxy)-14,19-dioxa-5,7,26-triaza-tetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8,10,12(27),16,21,23-decaene; Cell Therapeutics) is an orally bioavailable inhibitor of JAK2 and the JAK2 mutant JAK2V617F with antineoplastic activity. SB1518 competes with JAK2 for ATP binding, thereby inhibiting JAK2 activation. SB1518 is currently being evaluated in Phase I and I/II clinical trials in the US for treatment of chronic idiopathic MF as well as advanced lymphoid malignancies including malignant HL and B-cell lymphoma and advanced myeloid malignancies including AML, CMML and CML. A Phase I study of SB1518 has provided evidence of activity in patients with relapsed lymphoma. Patients were treated at doses up to 400 mg orally daily [82]. Eighteen patients were enrolled, and three patients at the 300-mg dose level demonstrated disease response (1 FL, 1 SLL and 1 MCL). Eleven patients (73%) had stable disease. The effect of drug treatment on p-JAK2, p-STAT3 and p-STAT5 was examined and SB1518 inhibited the JAK/STAT pathway as early as 4 h after administration.

1.1.9 Aurora kinase inhibitors

The Aurora kinases (AKs) have emerged as particularly promising targets due to their roles in regulating multiple signaling pathways crucial for accurate cell division.

The AKs have shown to be promising drug targets in pre-clinical models and several small molecules targeting Aurora kinases A and B or both have entered the clinic. Alisertib (MLN8237, 4-{[9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino}-2-methoxy-benzoic acid; Millennium Pharmaceuticals), a selective, ATP-competitive oral reversible AA inhibitor, is furthest along in the development. The peripheral T-cell lymphomas (PTCLs) are uncommon lymphoid diseases that account for 10–15% of all NHL cases in the US. Compared with B-cell NHLs, the PTCLs are generally aggressive and less responsive to current treatment options. Relapsed and refractory diseases are common. Novel and target therapies are in much need to improve quality and duration of response. In a Phase II trial of Alisertib administered orally twice daily for 7 days on 21-day cycles in patients (n = 48) with relapsed aggressive NHL including eight patients with PTCL, the ORR was 32% [83]. Response in T-cell NHL was the most impressive at 57%: four out of eight patients responded, and the some of the responses were durable. The ORR in DLBCL and MCL was modest around 20%. Treatment-related side effects were generally tolerable and included low blood counts, fever, fatigue and inflammation in mouth. Based on these results, a Phase III, open-label, multicenter trial is ongoing in patients with relapsed or refractory PTCL who will be randomized to Alisertib or investigator’s choice (Pralatrexate, Gemcitabine or Romidepsin) [84]. The primary endpoints are the ORR and PFS. Secondary endpoints include CR and unconfirmed CR, overall survival, time to progression and quality-of-life assessments.

Biomarkers of response are necessary to assess the degree of target inhibition in patients who are treated with aurora kinase inhibitors. Patient selection based on aurora A and/or aurora B overexpression may be a useful strategy to test whether these subpopulations will benefit from therapy with aurora kinase inhibitors. Some histologically aggressive lymphomas, for instance, are known to overexpress the aurora kinase A gene. The mitotic index constitutes a biomarker for Aurora A activity, because inhibition of Aurora A in cancer cells resulted in accumulation in mitosis [85]. Another biomarker for Aurora A activity is the degree of autophosphorylation on threonine at residue 288 (T288) of histone H3 [86]. Biological evidence of Aurora B inhibition is the inhibition of histone H3 phosphorylation on serine 10 [87]. Future studies with AK inhibitors are likely to be in combination with radiotherapy, chemotherapy or other targeted anticancer agents already in use for the treatment of various hematological cancers.