Patients with acute lymphoblastic leukemia (ALL) with t(4;11)(q21; q23)/MLL-AF4 are characterized with poor prognosis, aggressive clinical features and even with current treatment strategies, 5-year survival remains low (15 to 35 %) indicating the need for alternative therapies (Ghazavi et al, 2015; Mullighan, 2012; Marchesi et al, 2011; Sanjuan-Pla et al, 2015)(Ghazavi et al, 2015; Mullighan, 2012; Marchesi et al, 2011; Sanjuan-Pla et al, 2015). Panobinostat has shown promising effects in clinical trials with various types of neoplasms such as ALL (Giles et al, 2006)(Giles et al, 2006), melanoma, prostate cancer, breast, high grade gliomas, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia (Li et al, 2014)(Li et al, 2014). Additionally, in 2015, Panobinostat (Farydak®) was approved by FDA for treatment of refractory Multiple Myeloma (Laubach et al, 2015)(Laubach et al, 2015).
In vitro and in vivo studies have also shown promising antineoplastic effects for ALL therapy using Panobinostat alone and in combination with other chemotherapeutic agents such as vincristine and dexamethasone (Stumpel et al, 2012)(Stumpel et al, 2012). In the present study we evaluated the in vitro effects of the combination of LBH589 with MTX and 6MP in the RS4;11 cell line from an ALL adult patient harboring t(4;11). Surpringly, only the combination of Panobinostat 25nM + 6PM 50uM showed moderate synergism (CI = 0.78) and others combinations of Panobinostat with MTX and 6MP evaluated in this study showed antagonistic effects. We hypothesize that drug combinations improve therapy efficacy trought dose reduction, which leads to decrease in citotoxicity; however, it may promote antagonistic effects related to novel properties of the chemical mixture and biological interactions in ALL patients (Chou, 2006, 2010)(Chou, 2006, 2010).
The antagonistic effects of Panobinostat combinations with MTX and 6MP observed in this study may be related to specific properties and chemical interactions of these chemotherapeutics. 6MP is a purine analogue that is partially metabolized in the body and converted to the 6-thioguanine nucleotide by the action of various enzymes such as Hypoxanthine Guanine Phosphoribosyltransferase and Fosforibosyl Pyrophosphatase. 6MP is incorporated into DNA of nucleated cells, which identifyes the presence of the guanine analog, and activates repair systems that induce double-strand breaks and apoptosis (Schmiegelow et al, 2014)(Schmiegelow et al, 2014). MTX is a folate analogue that is converted to MTX polyglutamates that accumulate in the intracellular compartment by inhibiting the activity of enzymes that act on the synthesis of purines and pyrimidines, increasing the efficiency of the treatment. One of the enzymes involved in the conversion of MTX to MTX polyglutamate is Folilpoliglutamyl Transferase (Schmiegelow et al, 2014)(Schmiegelow et al, 2014). Considering that Panobinostat is a pan-inhibitor of HDACs and promotes pleiotropic effects in cells (Li et al, 2014)(Li et al, 2014), theoretically the drug could interfere with the activity of enzymes that control the intracellular metabolism of MTX and 6MP reducing the activity of these chemotherapeutics resulting in antagonistic effects on these combinations.
Some studies have also shown that MTX is structurally similar to some HDACi of the hydroxamic acid class (SAHA, TSA) and that MTX can bind to active sites of Histone deacetylase proteins (Yang et al, 2010)(Yang et al, 2010). Thus, it is also possible that the binding of MTX to HDACs may compete, interfere or preclude binding of Panobinostat, a hydroxamic acid, attenuating its effects on cells. However, other studies should be conducted to support these hypotheses.
Considering all these factors, and that for diseases such as cancer, high levels of synergy are much more relevant from the therapeutic point of view, the results of this work indicate that combinations of Panobinostat with MTX and 6MP may not represent an efficient strategy for the intervention therapy with ALL t(4;11). However, these results should be carefully considered as they reflect the effects of in vitro combinations on exposure times and specific drug concentrations and may not reflect the effects of combinations in vivo and its interactions with biology systems. We did not find other studies regarding the effects of the combination of Panobinostat with MTX and with 6MP.
Although it did not show synergistic effects in vitro when combined with MTX and 6MP in ALL with t(4;11) in this study, other studies have shown that Panobinostat acts synergistically when combined with other drugs (Vilas-Zornoza et al, 2012; Berdeja et al, 2021). Recently, Berdeja et al. (Berdeja et al, 2021) stablished that Panobinostat plus Carfilzomib is an effective steroid-sparing regimen for relapsed/refractory multiple myeloma in a phase I/II clinical trial study (Vilas-Zornoza et al, 2012). Additionally, Vilas-Zornoza and coworkers (Vilas-Zornoza et al, 2012) demonstrated that BALB/cA-Rag2 xenotransplanted mice with ALL cell lines TOM-1 (54-year-old woman LLA-B, Philadelphia +) and MOLT-4 (male ALL-19 relapse) showed significant reduction in tumor growth after treatment with Panobinostat (24 days, 5mg/kg). Panobinostat (5 mg/kg) also potentiated the effects of vincristine (0.025 mg/kg) and dexamethasone (1 mg/kg) in xenotransplanted mice with ALL primary cultures (from B and T cells origins), reduces the amount of blast cells in pheripherical blood, increases mice survival and promotes reduction of oncogenic protein CDK6 and phosphorilated RB (Vilas-Zornoza et al, 2012)(Vilas-Zornoza et al, 2012). However, the study by Vilas Zornoza and colleagues (Vilas-Zornoza et al, 2012)(Vilas-Zornoza et al, 2012) was not specific for ALL with t(4;11) which has peculiar molecular characteristics.
The t(4;11) ALL exhibits characteristic patterns of gene expression and DNA methylation that presumably favor the development of ALL. In silico studies using the cMap tool (Connectivity Map) have shown that iHDACs are compounds capable of reversing signatures of t(4;11) ALL, reduced expression of several hypomethylated (overexpressed) proto-oncogenes in ALL with t(4;11) and in vitro assays showed that Panobinostat also promotes reduction of oncogenic proteins MYC and RUNX1 (Stumpel et al, 2012)(Stumpel et al, 2012).
In addition, among several iHDACs (TSA, SAHA, VPA, FK228, MS275), Panobinostat showed preferential action on cell lines with t(4;11) (SEM and RS4;11). The drug also showed a greater effect in primary B-ALL cultures with t(4;11) (n=15) compared to primary B-ALL cultures without t(4;11) (n=6) and practically did not affect cultures of B cells from non-neoplastic bone marrow (Stumpel et al, 2012)(Stumpel et al, 2012). All these data indicate the need for in vivo studies on the effects of Panobinostat on ALL with t(4;11) for subsequent clinical screenings.
In the present study we confirmed the efficacy of Panobinostat in the in vivo model of ALL with t(4;11) and we observed that the drug administered in isolation promoted a reduction in the percentage of peripheral blood blasts and increased survival of the mice, suggesting a potential therapeutic strategy for ALL with t (4; 11). In vitro studies suggest that one of the major molecular mechanisms that influence the leukemogenesis process in ALL with t(4;11) is that the MLL fusion protein recruits an alternative histone methyltransferase called DOT1L (DOT1-Like Histone H3K79 methyltransferase) which in turn promotes histone methylation (H3K79me2) and induces increased expression of homeobox genes (HOXA7, HOXA9 HOXA10 and MEIS1 cofactor) resulting in increased proliferative capacity and autosterovation of blasts presumably favoring the development of leukemia (Chen & Armstrong, 2015; Ghazavi et al, 2015; Mullighan, 2012; Wong et al, 2007; Orlovsky et al, 2011)(Chen & Armstrong, 2015; Ghazavi et al, 2015; Mullighan, 2012; Wong et al, 2007; Orlovsky et al, 2011). However, in the present study, we did not observe differences in expression of homeobox genes (HOXA7, HOXA9, HOXA10 and MEIS1) between control and Panobinostat-treated mice, suggesting that other molecular mechanisms may be involved in the anti-neplastic effects of Panobinostat in ALL with t(4;11). In fact, some studies indicate that cell transformation in ALL with t(4; 11) (q21; q23) can occur through different pathways (de Braekeleer et al, 2012)(de Braekeleer et al, 2012).
Recently another work has described that Panobinostat promotes antineoplastic effects in murine models of infant ALL with rearrangements in MLL, increasing overall survival burden, and suggests that a molecular mechanism of action of the drug involves the suppression of the BRE1 Complex (RNF20/RNF40/WACE3) and H2B ubiquitination depletion (Garrido Castro et al, 2018)(Garrido Castro et al, 2018). In the study conducted by Castro et al. (Garrido Castro et al, 2018)(Garrido Castro et al, 2018), were used infant ALL cell lines from B-cell precursors, SEM (5-year-old girl relapsed; MLL/AF4), KOPN8 (3-month-old girl; MLL/ENL), and a primary culture of childhood ALL with rearrangement in MLL. The mice xenotransplanted with the cell lines and treated with 5mg/kg of LBH589 (n=7) had greater survival compared to the control group (n=6). In addition, none of the xenotransplanted mice with the primary culture of ALL (n=5) and treated with LBH589, succumbed to the disease demonstrating excellent results (Garrido Castro et al, 2018)(Garrido Castro et al, 2018).
It is important to note that Castro et al., worked with a model of infant/childhood ALL while in the present study, the cell line of ALL was used from an adult patient with t(4;11). Studies of gene expression profiles in ALL with t(4;11)(q21;q23) have identified that cases of adults and pediatric cases have different signatures, suggesting that different pathways of leukemogenesis may be involved (de Braekeleer et al, 2012)(de Braekeleer et al, 2012), reinforcing the idea that ALL in adults and children represent very different pathologies (Bueno et al, 2012, 2011; Sanjuan-Pla et al, 2015; Marchesi et al, 2011)(Bueno et al, 2012, 2011; Sanjuan-Pla et al, 2015; Marchesi et al, 2011) .
All these data indicate that Panobinostat has great therapeutic potential for childhood ALL with rearrangements in MLL that present markedly dysregulated epigenome, unfavorable prognosis and do not respond to conventional treatment. The results of the present study indicate that the drug also showed promising results in the ALL model of adults with t(4;11) and could represent an attractive chemotherapeutic for clinical screening. Further studies should be conducted to further elucidate the molecular mechanisms of action of the drug in vivo, and to evaluate the safety and efficacy for future use in clinical trials in ALL patients with t(4;11).