Studies targeting BTK have attracted substantial attention due to its crucial role in the BCR pathway and BTKi have shown promising antitumor activities, both in vivo and in vitro models. According to its mechanism of action and their binding affinity and activity, the BTKi are classified into two types: irreversible inhibitors, where they bind to the amino acid residue Cys481 forming a covalent bond; or the reversible inhibitors, which bind to an inactive conformation of the kinase, by accessing the specific SH3 pocket of BTK [58].

The majority of BTKi are irreversible inhibitors, from which the first-generation inhibitor is ibrutinib. In 2010, ibrutinib was demonstrated to have selective toxicity in DLBCL cell lines with chronically active BCR signaling with sub-nanomolar activity (IC₅₀ = 0.5 nM), by preventing the BTK autophosphorylation [40] and in vivo data confirmed its potential [59]. Ibrutinib, when combined with bortezomib, also presented a synergism by enhancing apoptosis in DLBCL and MCL cells through AKT and NFκB inactivation [60], while in DLBCL it showed cumulative antitumor effects with other agents, such as enzastaurin [61] or lenalidomide [62]. Together these preclinical studies provided detailed insights into the mechanism of action for the subsequent clinical trials (described below).

The off-target side effects of ibrutinib have led to the development of second-generation inhibitors of which acalabrutinib demonstrated to have less off-target kinases inhibition [63] and also to be more potent in in vitro assays and in vivo canine models of DLBCL and xenograft models derived from ABC-DLBCL and MCL [63,64,65]. Another more selective inhibitor is zanubrutinib, which also showed antitumor activity in the nanomolar range in MCL cell lines as well as in ABC-DLBCL cells, with a similar effect as ibrutinib but with less off-target effects and prolonged overall survival in a DLBCL xenograft model [66,67]. Alternative irreversible BTKi are M2951 and M7583, presenting an in vivo antitumor activity in preclinical models of ABC-DLBCL [68]; spebrutinib (CC-292), with high efficacy as a single agent as well as in synergistic combinations in ABC-DLBCL, but limited efficacy in GCB-DLBCL [69]; tirabrutinib (ONO/GS-4059), which is showing promising results in preclinical studies [70,71]; TG-1701, a more selective inhibitor, presenting Ikaros as an important biomarker for response and efficacy, both in vitro and in vivo [72]; and other compounds, currently under development with promising results in vitro, such as QL47 [73].

However, primary and secondary resistances have emerged from the irreversible BTKi ibrutinib, resulting in a poorer prognosis of relapsed lymphoma patients. In DLBCL and MCL the mechanisms of resistance are not as well-known as in CLL, where the cause of resistance has been identified to be related in part to mutations at the covalent site (C481) on BTK and also PLCγ2, resulting in a downstream signal activation [74].

To overcome the irreversible BTKi limitations (off-target side effects and long-term toxicities), new reversible compounds are under development that will be helpful for the ibrutinib-resistant patients. Some of the most relevant reversible inhibitors are ARQ-531, which have demonstrated potent activity against both BTK wild-type and C481S mutant in ABC- and GCB-DLBCL cell lines [75,76]; LFM-A13, a dual inhibitor against BTK and PLK [77]; HBW-3-10, with great potency and pharmacokinetic profile and better results than the ibrutinib, when compared in xenograft models [78]; fenebrutinib, a potent reversible inhibitor against BTK C4815 mutant [79], CG-806, with great effects in MCL and DLBCL in vitro and MCL PDX mice models [80], CB1763, with a strong effect on C481S mutant BTK [81]; and Pirtobrutinib (LOXO-305), with potent antitumor effects both in vitro and in vivo [82,83].

Another interesting novel strategy to overcome ibrutinib resistance is the proteolysis-targeting chimera (PROTAC), which will selectively target BTK C481S mutant. L18I has been reported to inhibit proliferation in BTK mutant DLBCL cell lines and induce rapid tumor regression in C481S BTK HBL-1 xenograft tumors [84]. In addition, P13I and compound 6e, are under development in preclinical studies with promising results [85,86,87,88].

The next key component of the BCR signaling pathway, which has gained relevance as a therapeutic target, is SYK. R406 is a SYK inhibitor that induces apoptosis in the majority of DLBCL cell lines studied by the inhibition of both tonic and induced BCR signaling [53]; however, it also inhibits other kinases [89]. Its oral prodrug is R788, fostamatinib, which has shown great efficiency in B-NHL-like mice models and is currently in clinical trials (see below) [90,91]. Another SYK inhibitor is cerdulatinib, which has demonstrated great antitumor activity by inducing apoptosis and cell cycle arrest in both ABC- and GCB-DLBCL cell lines [92]. Similar results were being observed for PRT060318, where the cell cycle arrest effect by the inhibitor was mimicked by a genetic reduction of SYK using a siRNA [89]. Entospletinib (GS-9973) selectively inhibits SYK and presents a synergistic antitumor effect with vincristine, both in a panel of DLBCL cell lines and in a DLBCL tumor xenograft model [93]. TAK-659 is a dual SYK/FLT-3 inhibitor with antitumor activity in DLBCL cell line xenograft models and in patient-derived xenografts (PDX) [94,95]. ASN002 is a dual JAK/SYK inhibitor, showing a great antiproliferative activity in in vitro and in in vivo models. Moreover, it also showed antitumor activity in ibrutinib-resistant cell lines [96].

Lastly, elevated or aberrant activation of mTOR has been identified in several cell lines and patient samples of DLBCL and MCL; thus, its targeting is a therapeutic approach alone or in combination [113]. Rapamycin, or sirolimus, is an antibiotic and the first mTOR inhibitor discovered, from which novel rapamycin analogs have emerged: temsirolimus that has shown antitumor activity in MCL cell lines [114]; and everolimus (RAD001), showing an effect associated with cell-cycle arrest in MCL [115]. To achieve a more potent anticancer activity, small molecules that inhibit both mTORC1 and mTORC2 have been developed. Among them, CC-223 is a potent and selective inhibitor that shows a better induction in apoptosis, when compared to rapamycin in a panel of DLBCL, FL, and MCL cell lines [116]. AZD014 has been shown to synergize with ibrutinib and cause tumor regression in in vivo experiments in ABC-DLBCL [117]. Finally, PQR309 is a dual PI3K/mTOR inhibitor with promise for advancing into clinical trials, alone or in combination with other treatments [118]. Figure 1 summarizes the main BCR signaling therapeutic targets in DLBCL and MCL.

Figure 1. Regulation of BCR signaling and the therapeutic inhibition of BTK and PI3K in DLBCL and MCL. (Left panel) Antigen-dependent and chronic active BCR signaling; (Right panel) Tonic BCR signaling.

The arrival of ibrutinib to the therapeutic armamentarium of MCL was a real breakthrough and has changed the treatment paradigm in the relapsed/refractory (R/R) setting. The first clinical results from the phase 2 pivotal study in heavily pre-treated MCL [119] led to an accelerated approval of ibrutinib for R/R MCL in 2013 by the U.S. Food and Drug Administration (FDA) and in 2014 by the European Medicines Agency (EMA). Ibrutinib showed an overall response (OR) and complete response (CR) rates of 68% and 21%, respectively, with a median duration of response (DOR) of 17.5 months, which were significantly higher than those observed with the previously approved targeted agents, bortezomib, temsirolimus, and lenalidomide, whose rates of OR and CR were 22–33% and 8–10%, respectively, with median DOR ranging between 8 months with bortezomib [120] and temsirolimus [121,122,123] and 16 months with lenalidomide [124,125]. The phase 3 RAY study demonstrated the superiority of ibrutinib versus temsirolimus also in terms of progression free survival (PFS) (15.6 vs. 6.2 months) [126]. In addition, patients receiving ibrutinib on their first relapse showed a significantly higher PFS than those treated with ibrutinib in later relapses (25.4 vs. 10.3 months, respectively), which was an unexpected finding, not observed in the temsirolimus arm, where the PFS was the same for all the patients regardless of the number of previous lines received. Another study pooling together the data from up to 330 patients with R/R MCL prospectively included in two phase 2 trials and in the phase 3 RAY study confirmed that the sooner ibrutinib is used when patients with MCL relapse after the first line, the better the results will be in terms of PFS, but also of CR rate (32% when ibrutinib was used after only one previous line versus 14% when used in later relapses), DOR (35.6 vs. 16.6 months with early vs. late use of ibrutinib) and overall survival (OS) (61.6 vs. 22.5 months) [127], leading to the current unanimous recommendation about the preferential use of ibrutinib as the first option in relapse, in order to get the most out of this active drug in R/R MCL.

Ibrutinib is a well-tolerated and safe drug, but the concern about its collateral effects on other kinases besides BTK, associated with a higher risk of bleeding (mostly grade 1–2 events in the clinical practice) and cardiovascular events (atrial fibrillation and hypertension) led to the development of new generation BTKi with reduced off-target effects. Acalabrutinib and zanubrutinib are the first two of this new generation BTKi with an improved safety profile [128,129] approved for patients with R/R MCL, with encouraging data on efficacy and survival confirming how this family of drugs has definitely changed the landscape of salvage treatment in MCL. Indeed, several newer, safer, and more potent BTKi are coming along, both covalent irreversible (such as acalabrutinib and zanubrutinib) and non-covalent reversible BTKi (such as pirtobrutinib and others), whose main clinical data are summarized in Table 1.

 

Inhibitors of mTOR (mTORi) and SYK (SYKi) have also been evaluated in MCL and DLBCL. The mTORi temsirolimus was approved in 2009 in Europe for R/R MCL based on its efficacy in two phase 2 studies, showing ORR of 38-41% in monotherapy [121,122], later confirmed in phase 3 studies, with ORR between 22-47% [123,126]. Regarding SYKi, fostamatinib showed modest activity in monotherapy in both MCL and DLBCL in a phase 1/2 study, with ORR of 11% and 22%, respectively [148], and, more recently, TAK-659, a dual inhibitor of SYK and FMS-like tyrosine kinase 3 (FLT3), showed ORR of 28% in patients with R/R DLBCL [152]. Studies evaluating these and other inhibitors of mTOR and SYK, both in monotherapy and in combination, are summarized in Table 1 and Table 2.

3. Conclusions and Perspectives

With the development of genomic sequencing and immunotherapy, the treatment of aggressive B-cell lymphoma entered the era of precision therapy almost two decades ago. Targeting BCR signaling with oral kinase inhibitors has changed the treatment landscape in MCL. In this disease, the introduction of BTK inhibitors represents one of the most important advances in small molecule-based therapies, with high response rates and durable remissions in the relapse setting, and very encouraging results in the frontline setting. Conversely, despite the initial excitement and strong biological rationale, the latter has yet to demonstrate significant improvements in the outcome for DLBCL patients. As an example, single-agent ibrutinib is active and well tolerated in non-GCB DLBCL, but the duration of response is remarkably short in unselected patients, compared to MCL. In addition, in DLBCL, these approaches are often associated with significant toxicities in combination with chemo-immunotherapeutic regimens, such as R-CHOP, probably underlying the biological complexity and aggressiveness of this disease. Thus, durable responses in DLBCL patients will undoubtedly require combination therapies targeting genetically and experimentally validated biologic drivers. In this sense, “chemo-free” combination strategies associating ibrutinib with rituximab and lenalidomide in previously untreated non-GCB cases are showing prolonged responses with some complete responses, suggesting that these new regimens may have a role in frontline therapy. However, longer follow-ups will be required to confirm these promising results.

Besides DLBCL, these combinations may also improve the clinical outcomes of MCL patients, while ensuring manageable toxicity. The most recent trials are taking advantage of the safety profile of second-generation PI3Ki or BTKi, associating them either with secondary agents with a distinct mechanism of action, such as the BCL-2 antagonist venetoclax, the cyclin-dependent kinase (CDK)4/6 inhibitor (palbociclib), or together (ibrutinib + umbralisib or ibrutinib + copanlisib), in order to enhance the blockade of BCR signaling.

Finally, the use of CAR-T and immune checkpoint blockade therapies, such as programmed cell death protein 1 (PD1)/PD1 ligand (PD-L1) antagonists in combination with BTK (and probably PI3K) inhibitors may represent a significant step towards tailored medicine for the clinical management of both MCL and DLBCL.