B-cell lymphomas
Lymphomas are the most common hematological malignancy in the United States and constitute about 5.4% of all cancers with a yearly estimated incidence of 66,000 cases of NHL and 5,000 cases of HL per year [
94]. B-cell NHL are the most common type of lymphomas and include subtypes that vary from some of the most aggressive tumors known to science (Burkitt’s lymphoma) to some of the most indolent (follicular and small lymphocytic). The lymphomas are readily classified into aggressive subtypes, for which urgent chemotherapy is required to affect cure, or indolent diseases, which are more typically managed as chronic diseases. As a result, treatment is often tailored to the aggressiveness of the disease, with dose intense combination therapy and possibly autologous bone marrow transplant being considered for aggressive histologies like with Burkett’s lymphoma, mantle cell lymphoma and diffuse large B- and T-cell lymphoma, while single agent rituximab and less dose intense chemotherapies are routinely employed for the more indolent subtypes of disease.
With this background, the development of new drugs for the treatment of lymphoma can be viewed as falling into one of several distinct categories: (1) simple single agent regimens that have a role in managing relapsed or refractory diseases (ex: bortezomib, pralatrexate for PTCL, vorinostat and romidepsin for CTCL, rituximab for indolent lymphomas); (2) integration of an active agent into an existing regimen to exploit possible synergistic effects (ex: rituximab plus CHOP or EPOCH); (3) use of the agent as a maintenance therapy following definitive chemotherapy (ex: rituximab, trials of lenalidomide in mantle cell lymphoma). In this context, each and every one of these opportunities exists across the many disease sub-types of NHL. Hence, there is a need to develop novel therapies for patients with NHL to continue to improve outcomes, which have clearly improved dramatically over the past decade. HDAC inhibitors, given their reasonable side effect profile and compelling effects on molecular pathways that may be important in lymphomagenesis, clearly have the potential to impact all these major areas of drug development. Presently, there are no HDACI agents that are approved specifically for the treatment of B-cell lymphomas, though there is data with select inhibitors, and a strong rationale remains intact for their use in B-cell malignancies. Mechanistically one disease where there appears to be a strong rationale, though with minimal clinical data to date, is in mantle cell lymphoma. This unique disease is grossly characterized by marked dysregulation of cyclin D1 mediated by the t(11:14) translocation, and loss of the cyclin dependent kinase inhibitors (cdk) p21 and p27. Interestingly, two of the prominent effects of HDAC inhibitors is down-regulation of cyclin D1, and up-regulation of p21/p27. The idea that one drug could potentially revert these pathognomonic molecular features represents a promising venue for thinking about how to rationally employ such agents for such a challenging disease. Similarly, the association of dysregulated BCL-6 in many cases of DLBCL and the effects of HDACI on BCL6 as described in the section above can potentially provide a rationale for the therapeutic opportunity.
Perhaps one of the earliest demonstrations of activity of an HDAC inhibitor in B-cell lymphoma was with one of the oldest HDAC inhibitors used in medicine, namely valproic acid (Depakote). Valproic acid, approved for the treatment of seizure disorders, induced a complete remission in a patient with multiply relapsed transformed follicular lymphoma, and was one of the first published reports of the efficacy of HDAC inhibitor in the treatment of B cell malignancies [
95]. Valproic acid, an acknowledged though weak inhibitor of histone deacetylases, has been more widely studied in myeloid leukemias, with little to no other experiences in lymphoid malignancies.
Beyond valproic acid, there have been several early phase trials with a variety of other HDAC inhibitors for the treatment of B-cell NHL. Kelly and O Connor [
78] reported on the first phase 1 study of intravenously administered vorinostat (previously known as suberoylanidie hydroxamic acid, or SAHA) in patients with solid tumors and hematological malignancies. Of the 11 patients with B-cell malignancies (HL
n = 5, NHL
n = 6 ), no patients with B-cell NHL responded, though antitumor activity was seen in two patients with HD including a 30% reduction of tumor burden in one patient which was maintained for 3 months and stable disease in another patient for 8 months. A follow-up Phase 1 study conducted by the same investigators [
83] with an oral formulation of vorinostat revealed a very favorable side-effect profile, and a maximum tolerated dose of 400 mg per a day given for 14 consecutive days on an every 21 day cycle. This study enrolled a total of 25 patients with hematological malignancies, with the best response being a complete remission (CR) seen in a patient with transformed follicular lymphoma. Partial responses (PRs) were seen in patients with transformed lymphoma and mycosis fungoides. A patient with Hodgkin’s disease had a 31% decrease in disease lasting for 10 months. These two studies of vorinostat in patients with solid tumor and hematologic malignancies were among the first to demonstrate the potential activity of these compounds in B-cell malignancies and Hodgkin lymphoma. Another Phase 1 trial of oral vorinostat has been conducted in patients with B-cell malignancies, as recently reported by investigators in Japan. This trial enrolled ten patients with B cell lymphomas at two dosing levels: 100 mg and 200 mg bid for 14/21 days. Four of the ten patients responded, including two CRs and one PR seen in patients with follicular lymphoma (FL) and one PR in a patient with MCL [
96]. A dedicated phase 2 study of vorinostat in patients with relapsed DLBCL [
97] at a dose of 300 mg bid (14 days per 3 weeks or 3 days per week) has been reported. Eighteen patients were enrolled, with one patient obtaining a CR lasting 468 days, and one patient who attained stable disease for 301 days. Based on this study, and the collective experience from the Phase 1 studies, it was concluded that vorinostat did not exhibit impressive single agent activity in large B-cell lymphoma. However, it should be noted, that the MTD now part of the label for vorinostat, was not used to assess response in these studies, and in fact, based on the phase 1 experience, as well as data generated by Duvic et al. in CTCL, this particular dose was found to be poorly tolerated. A second phase II study evaluated vorinostat at a dose of 200 mg bid for 14/21 days in patients with relapsed and refractory indolent NHL. This trial enrolled 33 out of which four patients achieved a CR, two attained a PR and 4 had stable disease [
98]. These experiences seem to suggest that while the single agent activity of vorinostat in DLBCL is variable, and probably low, it raises two question: (1) why do select patients can achieve CR, while other don’t; and (2) what is the optimal pharmacokinetic profile.
The experience with vorinostat has prompted further evaluation of other more potent HDAC inhibitors for the treatment of lymphomas. Parallel to the development of these agents in myeloid malignancies, most phase I trials are conducted to include the broad category of lymphoid diagnoses that initially encompass both B- and T-cells with the idea of expanding the trial to a specific subtype if a positive signal is seen in a specific diagnostic group. Belinostat, another HDAC inhibitor that belongs to the class of hydroxamic acids is currently in development for the treatment of lymphoma, in particular peripheral T-cell lymphoma (PTCL). Similar to vorinostat, it has an oral and IV formulation, though the majority of data collected to date is with the IV formulation. The initial phase I trial of belinostat in advanced solid tumors established a maximal tolerated dose (MTD) of 1,000 mg/m2 given IV for 5 days as a 30 min infusion. This dose was confirmed as the MTD in a parallel study of the same agent in hematological malignancies with no additional toxicity seen in this group of patients. This study enrolled 11 patients (DLBCL
n = 7, transformed CLL
n = 2, transformed FL
n = 4, CLL
n = 2). No objective responses were seen in any patients though some patients experienced stabilization of disease (DLBCL
n = 2, CLL
n = 3). For ease of administration, the oral formulation of belinostat is being developed and a Phase 1 study of oral belinostat in patients with all sub-types of NHL is underway. The starting dose was 750 mg/m2 with planned escalations until MTD is reached. Interim results on nine patients were reported at ASCO 2009 [
99]. Tumor shrinkage in the range of 43% to 49% was reported in one patient with MCL and two patients with Hodgkin lymphoma. Accrual continues as MTD has not been reached with present cohorts being treated at doses well above the MTD reached with the IV formulation again alluding to the differing properties of oral and IV forms of these agents.
Another orally administered HDAC inhibitor PCI-2478, belonging to the class of hydroxamic acid is being developed for the treatment of lymphomas. Evans et al have reported the results of the initial phase I data at ASH 2010. One patient with follicular lymphoma achieved a CR [
100] while four PRs and seven SD responses were noted in 20 evaluable patients. This study has shown that there are no cardiac effects or prolongation of the corrected QT interval (QTc) interval noted with this agent. A phase II portion of the trial is planned. Several other HDACI in development for various lymphoid malignancies are listed in Table
3.
While the experience with HDAC inhibitors as single agents in B-cell lymphoma has been less than hoped for, it is clear these drugs have important potential activity, and that the likely path forward will involve rational combinations. In addition, the experience with all the available HDAC inhibitors is not consistent, as some have minimal to no data in B-cell malignancies. It will be important to gain a better understanding of how the various classes of HDAC inhibitors work in B-cell lymphoma, and to try and develop some reasonable hypotheses around how these agents are likely working in these DLBCL. With this experience intact, the emergence of potentially important HDAC inhibitor combinations will emerge. As we will discuss below, understanding the molecular pharmacology in the context of understanding the molecular pathogenesis also highlights logical drug: drug combinations likely to be synergistic in particular disease settings.
Hodgkin lymphoma
Hodgkin’s lymphoma (HL) carries a cure rate of over 85% with either primary therapy or salvage therapy with high dose chemotherapy and stem cell transplantation. Patients who fail these first and second line regimens typically have a poor prognosis with a median survival of 3 years [
101]. While there appear to be many drug emerging for the treatment of NHL, it has been less obvious how any of these agents might perform in patients with relapsed or refractory Hodgkin lymphoma. To date, several HDAC inhibitors have shown activity in relapsed HL, and after T-cell lymphoma, it is the one disease venue where the clinical data is compelling enough to now entice some sponsors to pursue registration directed studies in HL.
Many lines of in vitro data support the potential activity of several HDACI against HL cell lines. HL is characterized by a high level of cytokine secretion from the inflammatory infiltrate surrounding the pathognomic Reed Sternberg (RS) cells. These cytokines include, IL-5, 6,7,9,10,13 and thymus activation-regulated chemokine (TARC/CCL17). These cytokines, many of which are part of an autocrine loop are thought to function by activating the JAK/STAT pathways, resulting in continual activation of the STAT family of transcription proteins. In particular, STAT3 and STAT6 are known to be frequently activated in HL. STAT3 activation is induced by several cytokines including IL-2, 6, 7, 9, 10,15 , while STAT6 is induced primarily by IL4 and IL13—and may be dependent on an autocrine IL-13 loop secreted by Reed Sternberg cells [
102]. Phosphorylated STAT6 localizes to the nucleus and induces the expression of STAT6 target genes that include TARC and IL-13 as well as other cytokines that attract lymphocytes that belong to the Th2 class into the tumor microenvironment [
103‐
105]. These lymphocytes are involved in humoral immunity and promote allergic responses. This suggests the importance of STAT 6 in the survival of Reed Sternberg cells as well as promoting the unique cellular and immunologic milieu that is a hallmark of HD. Interestingly, STAT regulation involves phosphorylation as well as lysine acetylation, which implies that HDAC inhibitors could play a role in regulating critical features of the HL biology [
106]. This is supported by the following evidence. Buglio et al [
107] have demonstrated that exposure of HL cells (L-428 and KM-H2) to vorinostat showed an increase in histone acetylation and p21 expression, and caspase mediated apoptosis. Vorinostat selectively inhibited STAT6 phosphorylation and was shown to result in decreased mRNA levels of STAT6 by PCR and a reduction of TARC as a downstream effect. Changes in cytokines were evaluated in the supernatants of exposed cells which demonstrated an increase in the level of IL13 and IP-10 (interferon inducible protein), and a significant decrease in the level of IL-5, confirming a shift in the Th1/Th2 cytokine balance. Another target gene regulated by STAT6 is the antiapoptotic Bcl-xL, which was shown to be significantly decreased in the HL cell lines following vorinostat exposure. This reduced level of Bcl-xl could alone reduce the apoptotic threshold sufficient enough to allow for rational combinations with other HL active agents. In addition to these effects on growth and survival pathways, it is also clear that HDAC inhibitors, either alone or in combination with a hypomethylating agent, can also influence antitumor immune responses by affecting the expression of proteins like the cancer testis antigens (CTA), which include MAGE, SSX and NY-ESO, in a variety of tumors including HL [
108]. These immunomodulatory effects may also contribute to the therapeutic activity of these agents in HL.
Clinically, there are now a several studies that seem to consistently demonstrate that HDAC inhibitors have activity in Hodgkin’s lymphoma. One of the earliest insights into this signal was revealed in the phase 1 experience with vorinostat. In the IV and oral phase 1 experiences with vorinostat, 12 patients with relapsed or refractory HD were treated with escalating doses of vorinostat, with four patients exhibiting responses as follows: On the IV study, one patient attained a PR that lasted for approximately 9 months, one patient experienced a 14% decrease in her lung disease resulting in significant improvement of her performance status, and a third patient achieved a 42% reduction of the tumor lasting for 2 months. On the oral study, one patient achieved a 31% decrease in tumor lasting nearly 10 months A subsequent phase II trial of vorinostat administered at 200 mg orally given twice a day for 14 of 21 days produced only modest clinical activity, with only one patient achieving a PR [
109]. It should be appreciated that the phase 1 experience with oral vorinostat established a MTD of 400 mg given orally once a day 14 of 21 consecutive days. This in fact is the recommended dose and schedule in the label of vorinostat for use in patients with relapsed or refractory CTCL. In fact, as we will see shortly, a well performed systematic analysis of different doses and schedules of vorinostat in CTCL clearly reaffirmed this MTD, and also provided data that alternative doses and schedules were less likely to produce clinical activity. Most, if not all phase 2 experiences with vorinostat in NHL, beyond CTCL of course, have been performed with alternative doses and schedules of vorinostat, raising an important issue: phase 2 studies employing schedules other than 400 mg PO daily may be using suboptimal dosing schedules, and thus, poor evidence in these phase 2 studies could reasonably be attributed not merely to the ‘activity’ of vorinostat in that context, but rather, to the dosing schedule. It will be important in future studies of vorinostat to employ at least the recommended phase 2 dose and schedule when exploring activity in different disease scenarios.
Of the available HDAC inhibitors, panobinostat is being studied in a registration directed phase 2 trial in Hodgkin lymphoma. The initial phase IA/II trial of panobinostat (LBH589) employed 2 different dose levels and schedules of this agent in patients with hematological malignancies. Patients with HL were entered onto the study at two dose levels: Arm 1 was dosed at a starting dose of 30 mg a day given Monday, Wednesday and Friday (MWF) every week, while arm 2 was initiated at 45 mg a day given on the same MWF every other week schedule. There were 13 patients with HL that were enrolled on the study, of which five met criteria for a partial response indicating evidence of activity HL. It appeared to be well tolerated with fatigue, nausea, thrombocytopenia and diarrhea being the most common side effects. The MTD was estimated by the logistic regression model and was defined as 40 mg a day given every MWF on the weekly schedule [
110,
111]. Based on this data, a large international phase II trial of panobinostat is designed for patients with relapsed and refractory HL. While the study is now on going interim results were reported on 81 patients at ASCO 2010 with responses seen in 17 patients, two patients attaining a CR and 15 patients attaining a PR, despite the fact that the study population appears to be very heavily treated [
112,
113].
Another HDAC inhibitor with activity in Hodgkin’s lymphoma is MGCD0103, which belongs to the benzamide class. MGCD 0103 is classified as an isotype selective HDAC inhibitor that predominantly inhibits class 1 HDAC enzymes and is administered as an oral formulation. A phase II trial of MGCD0103 was conducted at a dose level of 110 mg given orally three times per week in patients with relapsed and refractory HL. Responses were seen in seven of the 20 patients that were treated at this dose level including complete remissions; however the dose was considered too toxic, requiring frequent interruptions and dose reductions. The protocol was then revised to lower the dose to 85 mg per day give on the same schedule. Another ten patients were enrolled on the lower dose and partial responses were seen in three of the ten patients. The agent was well tolerated with the main side effects being fatigue and thrombocytopenia, though two patients developed pericardial effusions requiring an interruption of the protocol [
114]. Collectively, these data suggest that there is a signal of efficacy of some of the more potent HDAC inhibitors in HL but the biological correlates of this activity are still unclear.
T cell lymphomas
Mature T-cell non-Hodgkin Lymphoma and NK-cell neoplasm’s comprise about 12% of all NHL and 15–20% of aggressive lymphomas worldwide. They are characterized by great morphological diversity and genetic variation even within individual disease entities. The current 2008 WHO classification recognizes over 20 types of mature T-cell and NK T-cell lymphomas (PTCL) [
115]. Cutaneous T cell lymphomas (CTCL) are malignancies that arise in the skin and are classified as a separate entity based on their distinct clinical behavior and prognosis [
116]. Among the aggressive lymphomas, a T-cell phenotype confers a worse clinical outcome compared to their B-cell counterparts, with the exception of ALK-positive ALCL. Long-term survival at 5 years remains at 10–30% for most histologies with present treatment strategies [
117] and relapsed and refractory disease remains a significant clinical dilemma.
For reasons that are not entirely clear, HDAC inhibitors have shown relatively consistent and promising activity in the treatment of many types of T-cell lymphoma. In fact, two agents of this class, vorinostat (Zolinza) and romidepsin (Istodax) have been approved for the treatment of relapsed or refractory CTCL in the US. In addition, romidepsin, belinostat and panobinostat are all in clinical trials for T-cell malignancies, including peripheral T-cell lymphoma.
From a mechanistic perspective, it has been difficult to assign a precise mode of action of this class of drugs to any lymphoma, let alone CTCL or PTCL. It is not even clear if histone acetylation is essential for the biological activity seen in T-cell lymphomas. Pharmacodynamic studies have shown that histone acetylation can be demonstrated in peripheral blood mononuclear cells as well as [
83] tumor tissue from patients with T-cell lymphoma following treatment with HDAC inhibitors, however, there is no correlation with clinical response to these agents [
83]. Essentially every patient achieves acetylation of histone. Duvic et al. [
118] have attempted to look at biological correlatives of HDAC inhibitor therapy in patients with CTCL by performing serial skin biopsies on patients receiving vorinostat on trial at 2 h, and then at 4, 8 and 12 weeks after initiation of treatment. These results established the following: at 4 weeks, 39% of the patient samples demonstrated lymphocyte depletion consistent with the anti-lymphoma effect of vorinostat. Samples were also studied for acetylated histones at baseline and then at 4 weeks post therapy. Increased acetylation within lymphocytes post-therapy could not be demonstrated or correlated with response as most of the lymphocytes were not present after treatment. At 4 weeks post-therapy, there was a decrease in dermal microvessel density as measured by CD31 positivity on dermal vessels in all patients which was significantly lower in responding patients (
p = 0.001). Prior cell line data using the CTCL cell line HH indicated that a 24 h exposure to vorinostat resulted in an 8-fold increase of the antiangiogenic protein TSP-1 as studied by gene expression array, Consistent with the cell line data, an increase in the dermal TSP-1 staining was noted at 2 h in four out of eight patient samples including two responders, and at 8 weeks a dermal increase in TSP-1 was demonstrated in six of the 17 paired lesions including four of the six responders. Another important protein that is constitutively activated in CTCL is p-STAT3, which can be detected by immunohistochemical stains either in the nucleus or cytoplasm within both the keratinocytes and the lymphocytes present within lesions. In this study, nuclear staining for p-STAT-3 was prominent in both keratinocytes and lymphocytes prior to the start of therapy. After 4 weeks of therapy with vorinostat, the staining pattern shifted to localization within the cytoplasm in nine of the 11 patients who responded, where as this shift was noted in only three of the 16 non-responders. This shift was noted as early as 2 h after treatment in four of 11 paired lesions [
59].
Using a similar model of paired skin biopsy, Prince et al performed gene expression profiles (GEP) and real time quantitative PCR on skin samples from six patients with cutaneous T cell lymphoma who were being treated with panobinostat in a phase I trial. These biopsies were obtained at 0, 4, 8 and 24 h after administration of drug. In this study there were ten patients with a diagnosis of relapsed CTCL who were treated at varying dose levels as part of a large phase I study in patients with hematological malignancies. One patient received drug at 30 mg a day at the MWF weekly schedule and the remaining nine patients received panobinostat at 20 mg a day on MWF weekly. Clinical efficacy was observed in eight patients (two achieved a CR at both dose level and four patients achieved a PR, two patients had stable disease (SD)). The skin biopsy data demonstrated that there was hyperacetylation of histone H3 observed in tumor cells as early as 4 h after treatment. Consistent with previous data, histone acetylation within mononuclear cells was demonstrated in both responders and non-responders up to 48 to 72 h after the last oral dose indicating that this could not be used as a therapeutic marker. GEP data from all six patients consistently showed that panobinostat induced transcriptional regression of a greater number of genes than activation. Further statistical analysis indicated that 20 genes were consistently repressed and three genes were consistently activated following treatment with panobinostat. The genes that were consistently affected included genes affecting cell cycle (CCNDI, IGFI) apoptosis (septin10, TEF, SORBBS2), angiogenesis (GUCY1A1, ANGPT1) and immune modulation (LAIR1). Interestingly, CDKN1A, which codes for p21, has been shown to be consistently unregulated in response to HDAC inhibitor therapy, though in this study upregulation of p21 was not consistently seen in all patients. Of the 23 genes, 4 were further selected for validation by QRT-PCR. These data confirmed downregulation of guanylate cyclase 1A3 (GUCY1A3), the proangiogenic gene ANGPT1a and the transcription factor COUP-TFII (NR2F20 which is an upstream regulator of ANGPT1 and CCND1). This affect on genes controlling angiogenesis is consistent with the effects noted by Duvic et al. [
59] {as discussed above and provides confirmation that the anti-angiogenic affects of HDAC inhibitor therapy may be important in their mechanism of action. Bates et al. [
119] have conducted a clinical trial of romidepsin in patients with PTCL and CTCL and have also looked at the biological correlates of activity of HDAC inhibitor therapy. Predetermined markers of HDAC inhibitor activity including global histone acetylation were studied in peripheral blood sample. In addition tumor samples were collected to look for ABCB1 gene (encodes for the p-glycoprotein MDR) expression and an increase in the level of fetal hemoglobin as these genes were known to be modified by HDAC inhibitors therapy. The histone acetylation data correlated with PK parameters of AUC and C max, though there was no correlation between response, histone acetylation and the expression of either ABCB1 or fetal hemoglobin.
Increasingly, T-cell malignancies are found to be associated with dysregulation of the T-cell receptor (TCR) signaling and the immune function which contributes to the clinical syndrome. Hence, it is likely that the therapeutic effects of HDACI in T-cell lymphomas may be related to their effects on TCR and the associated immune effects. Investigators have started to look at the effects of HDAC inhibitors particularly vorinostat on TCR signaling and the immune system in order to delineate a more specific mechanisms of action for this agent, as well as to understand the basis for combining it with other agents. Woznial et al. [
120] have performed extensive studies using GEP on a panel of CTCL cell lines (HH, HUT78, MJ, Myla, SeAx) that were exposed to vorinostat at various time points. Their results show that as previously seen vorinostat exposure resulted in apoptosis in these cell lines in a dose dependent manner, affected cell cycle progression (G2M and G0/G1 arrest) and induced hyperacetylation of all four core histone proteins. GEP confirmed that 20% of the genes were significantly activated or repressed in response to vorinostat in 24 h. The functional analysis of these altered genes revealed pathways that have already been identified as being affected by HDAC inhibitors including cycle regulators for G1/S transition (E2F, E2F4, CDK4, CDk6, Cyclin A2, D2,D3,E20) ,G2/M regulators (CDC23, CD25B and CHEK4), apoptosis (FAS, IRAK1, CASP6, BID, BCL2), antiproliferative genes as well as multiple mitogen activated signaling kinase (MAPK) signaling pathway ( MAPK1, MAP3K6, MAP3K14) as described in the sections above. However, this study has demonstrated changes in genes that are involved in the JAK/STAT signaling pathway, cytokine–cytokine interaction and expression of receptors belonging to the tumor necrosis factor (TNF) family, all important pathways for survival and differentiation of lymphocytes and the immune system. Vorinostat treatment was shown to shift the expression profile of cytokines resulting in increased expression of interleukins like IL1a, IL6, Il9, and a decrease in the expression of IL4, IL5, Il10, IL11 and their associated receptors. Overall the cytokine profile represented a state that inhibited lymphocyte growth and proliferation and inhibited the TH2 type immune responses. This latter aspect of the drug effect is important as CTCL is a malignancy of activated T cells and is characterized by dysregulation of the immune system with reversal of the Th1/Th2 cytokine profile [
121]. Vorinostat has also been shown to affect genes that affect cell migration and chemotaxis that may affect the skin homing properties of malignant cells in CTCL. While it is difficult to list all the genes that were altered, overall there was a decrease in the expression of certain cytokine genes like CCL1, CCL22, CXCL10, CCR4 and CCR6 and an increase in others like CCR2, CCR6. There were also some alteration in the expression of members of the JAK/STAT pathway themselves like STAT6, STAT5A, SOCS2, (decrease) and STAT1, STAT3, JAK1 (increase).
Another important pathway required for T-cell survival and proliferation involves the T cell receptor (TCR). TCR signaling induces activation of many protein tyrosine kinases resulting in phosphorylation of many downstream substrates including CD3 chains, TCR epsilon chain, and the zeta chain associated with ZAP-70 and phospholipase C [
122]. After antigen stimulation, TCR activation leads to activation of LCK and FYN which results in tyrosine phosphorylation, after which ZAP-70 tyrosine kinase is recruited to the TCR where it acts together with LCK to activate downstream substrates including PKC, and MAPK as well as the Jun pathway. In addition, it is linked to the PI3K/AKT pathway which is important for the survival of T-lymphocytes through an increased expression of BCL-XL. Treatment with vorinostat induces repression of all genes associated with TCR related signaling including ZAP70, CD3DIL4, IL5, Il10, FOXP3 and upregulated FYN, IFNG and IL12A. The effects on TCR signaling were significant and were seen across all cell lines and confirmed by QRT-PCR. A decrease in the phosphorylated forms of ZAP-70 and AKT after vorinostat treatment was confirmed by western blots confirming the inhibitory effects of this agent on TCR signaling. FYN which seems to be upregulated by vorinostat functions is a SRC family kinase that phosphorylates several negative regulators of TCR signaling and ultimately adds to the negative effect of vorinostat on TCR signaling pathways. These changes were seen in cell lines and it is possible that there may be differences in gene expression that may be seen in actual tissue samples, and variability associated with the various subtypes of HDACI.
Clinically, there are many HDAC inhibitors that are being studied for the treatment of T-cell lymphoproliferative malignancies. While each has its own strengths and limitations, we will briefly review those HDAC inhibitors presently in clinical study for T-cell NHL.
Vorinostat (formerly known as suberoylanilide hydroxamic acid—SAHA) was the first HDAC inhibitor approved for the treatment of cancer. In the US this agent is approved for the treatment of CTCL in patients who have failed at least two prior systemic therapies [
1]. Based on early signals of activity and a favorable toxicity profile in patients with lymphoma in the phase 1 experience, Duvic et al. [
59] conducted a restricted phase I/II study of oral Vorinostat in patients with CTCL. This Phase 2 experience enrolled 33 patients with advanced heavily pretreated CTCL patients. Three different orally administered dosages and schedules were sequentially evaluated: 400 mg daily; 300 mg twice daily for 3 days followed by 4 days of rest; and 300 mg twice daily for 14 days with a week of rest followed by 200 mg twice daily. The overall response rate (ORR) was 24.4% with eight patients having a partial remission (PR) including four patients with Sezary syndrome. More importantly, 14 of the 33 patients (42%) reported significant pruritis relief. The median time to response was 11.9 weeks, and the median duration of response was 15.1 weeks. The major side effects included fatigue, thrombocytopenia, and gastrointestinal symptoms (predominantly diarrhea and nausea). Within the different cohorts, more thrombocytopenia was noted in the third cohort where patients received a higher dose of 300 mg bid for 14 days straight. However, most of the responses were seen in Groups 1 and 3. Thus, based on the response rate and toxicity criteria, the 400 mg per day dose was established as having the best safety profile. This study affirmed the findings in the original phase 1 experience, supporting the 400 mg PO daily dosing of vorinostat, which was associated with the most favorable side effect profile and highest response rate of any of the explored schedules. It is for this reason that conclusions about the efficacy of vorinostat with other doses and schedules may be misleading if the phase 2 study was not conducted at the recommended Phase 2 dose based on the Phase 1 experiences. Based on these data, a registration directed phase II study using the 400 mg PO dose was initiated by Olsen et al. [
93]. This study included 74 pts with CTCL who had failed at least two prior systemic therapies. Disease assessment was done by modified skin weighted assessment tools (m-SWAT). This study demonstrated an overall response rate (ORR) of 29.7%, with one patient achieving CR after 281 days of therapy. Median time to objective response was 56 days (28–171) though some patients took up to 6 months to respond and the median duration of response was not reached in the study though it was greater than 185 days. Non-progressing patients continued on the study with 15 patients receiving drug for over a year and six patients who received it for more than 2 years. The response to vorinostat was not impacted by their response to last systemic therapy. In addition, another 29 patients had clinical benefit manifested by stable disease for over 24 weeks. Prurtis relief was seen in 305 of patients including responders and non-responders again pointing to the effect of vorinostat on cytokine profiles. These data supported the approval of vorinostat by the U.S. FDA in October of 2006, making it the first HDAC inhibitor ever approved for the treatment of cancer.
Romidepsin (formerly referred to as depsipeptide, FK228) is cyclic peptide originally isolated from the broth culture of
Chromobacterium violaceum. It was initially studied at the National Cancer Institute in patients with refractory or relapsed solid tumors by Bates and Piekarz. Piekarz et al reported a case of patient with refractory PTCL that responded to depsipeptide [
123]. As the drug began to demonstrate a consistent signal in patients with T-cell lymphoma, the study was expanded to separate out patient with CTCL from those with PTCL, and then further stratified patients based on the amount of prior therapy they received. Overall it had a stratification that consisted of seven different treatment arms with the intention of separating out different cohorts of patients with T-cell lymphomas based on histology and prior number of therapies. All patients received drug as an intravenous infusion at a dose of 14 mg/m2 once a week in a 23 out of a 4 week cycle. The two major cohorts were CTCL and PTCL and the data from these two groups of patients has been analyzed and presented separately. Piekarz et al reported the results of 71 patients with relapsed CTCL that were treated with depsipeptide. The overall response rate was 34% (24/71) with complete responses observed in four patients. The median duration of response was 13.7 months. Based on the data generated by the NCI, the drug was eventually acquired by Gloucester Pharmaceuticals, and two registration directed clinical trials were launched, one in CTCL and one in PTCL. In the preparation of the New Drug Application to the U.S. FDA, data from the two studies (NCI plus Gloucester) was pooled and patients were analyzed using a composite end point to assess response using skin assessment, lymph node and visceral involvement and abnormal circulating Sezary cells. One-hundred thirty five patients were evaluable with a median age of 57 years who had received a median of at least 2 (1–8) prior systemic therapies. One hundred three patients (76%) had at least stage IIB or higher disease and 19 patients had SS. The overall response rate (ORR) was 41% with a complete response (CR) rate of 7% and duration of response of 14.9 months. Interestingly, the response rate (RR) was 58% in patients with Sezary Syndrome. Unique to the romidepsin study was a specific symptom assessment tool to quantify changes in pruritis. In this study a relief of pruritis was seen in over 60% of the patients. This agent was approved by the FDA for the treatment of relapsed CTCL following failure of one line of systemic therapy. This trial included cohorts of patients with PTCL who had failed at least one prior systemic therapy. The NCI sponsored study with 48 evaluable patients showed an overall response rate of 31% with 4 (8%) CRs. Patients who got more than 2 cycles of therapy had a response rate of 44%. Median Duration of response was 9 months with a median time to progression of 12 months. The results of the Gloucester sponsored study of romidepsin in patients with PTCL are still awaited.
Belinostat is a hydroxamic acid with pan HDACI activity that is currently in clinical trials for solid tumors and hematological malignancies available in both oral and intravenous formulation. A Phase I trial of the intravenous formulation was carried out in parallel to the solid tumor phase and an MTD of 1,000 mg/m2 was established. This formulation is being studied in a large phase II trial at a dose of 1,000 mg/m2 for patients with relapsed PTCL. At the same time the oral formulation has been developed and similar to the story with vorinostat has been shown to be better tolerated than the IV formulation. In the ongoing phase I trial of belinostat in hematological malignancies, the dose is up to 2,000 mg/m2 and an MTD is not reached [
124].
Oral Panobinostat, also known as LBH589, is a pan HDAC inhibitor belonging to the hydroxamic acid group that has shown remarkable activity in patients with CTCL in a phase I/II trial. Six out of ten patients with CTCL showed a response in the original phase 1 trial at a dose of 20 mg and 30 mg MWF on a weekly basis. The trial has now been expanded to a dedicated phase II in patients with CTCL at a dose of 20 mg a day MWF given weekly. The results of this trial are awaited.