Bruton's Tyrosine Kinase (BTK) Degrader Nx-2127 Exhibits Lethal Activity and Synergy with Venetoclax and BET Protein Inhibitor Against MCL Cells Sensitive or Resistant to Covalent BTK Inhibitors

Abstract

Bruton's tyrosine kinase (BTK) is a Tec family member, non-receptor, tyrosine kinase, which is constitutively active in Mantle Cell Lymphoma (MCL) cells, downstream of B cell receptor (BCR), Toll like receptors (TLR) and Fc receptors. BTK plays a critical role in cell cycle progression and B cell activation. In murine models, loss of function and kinase-dead mutants of BTK show impaired B cell maturation. BTK activates multiple pro-growth and pro-survival mechanisms, involving PLCγ2 activity, RAS-RAF-MEK-ERK pathway and NFκB transcriptional activity. Covalent BTK inhibitors (BTKis), including ibrutinib and acalabrutinib, irreversibly bind to cysteine 481 in the kinase domain of BTK, inhibit growth, and induce loss of viability of MCL cells. Although treatment with covalent BTKi induces dramatic clinical remissions, intrinsic and acquired resistance to BTKis is observed in majority of patients. BTKi-resistance in MCL cells has been attributed to activation of the alternative NFκB signaling due to TRAF2, BIRC3 or MAP3K14 mutation, gain-of-function CARD11 mutation, CCND1 mutation and MYC activation. Emergence of C481S BTK mutation causes steric hindrance to the covalent BTKi to C481, leading to acquired resistance, followed by clinical relapse in CLL, and less commonly in MCL. However, this situation still underscores dependence on BTK signaling for growth and survival of MCL cells. It also supports the strategy of using a BTK degrader for degrading and depleting BTK, including C481S-BTK, in MCL cells. NX-2127 and NX-5948 are promising, cereblon-based, orally bioavailable, clinical grade BTK degraders, which degrade and deplete BTK levels. In the present studies, we determined that in vitro exposure of human MCL cells, including MCL cell lines: REC1, Mino and JeKo-1, as well as patient-derived (PD) MCL cells, to NX-2127 (10 to 250 nM) for 2 to 24 hours markedly depleted BTK levels via proteasomal degradation, since co-treatment with the proteasome inhibitor carfilzomib restored BTK levels. NX-2127 but not NX-5948 treatment also degraded and depleted IKZF1 and IKZF3 levels. However, both BTK degraders also depleted BTK levels in BTK1-resistant Mino-IR and Jeko1-IR cells (which display induced in vitro resistance after recovery from repeated exposures to high levels of ibrutinib, i.e., 30 μM for 48 hours). NX-2127 treatment reduced the mRNA levels of IL-10 and TNFα in the MCL cells. Additionally, NX-2127 mediated depletion of BTK was associated with downstream reduction in the protein levels of p-PLCγ2 (Y1217), p-AKT, pERK1/2 and SOX11 levels in Mino, Mino-IR as well as in PD MCL cells. Notably, NX-2127 treatment also markedly depleted the protein levels of C481S-BTK that confers clinical resistance to covalent BTKis in CLL cells. Exposure to NX-2127 dose-dependently inhibited growth and survival of REC1, Mino, JeKo-1, Mino-IR, JeKo-1-IR cells, as well as of PD MCL cells. We have previously reported that BET protein inhibitor and/or BCL2 inhibitor (venetoclax) induce in vitro lethality in cellular models of MCL (Blood, 126: 1565, 2015). Based on this, we determined the synergistic lethal activity of NX-2127 and venetoclax or the BD2-selective, BET protein inhibitor ABBV-744 in MCL cells. Co-treatment with NX-2127 (10 to 100 nM) and low nM levels of venetoclax or ABBV-744 was synergistically lethal against MCL cells (Delta synergy score above 1.0 by ZIP method). Collectively, these findings demonstrate that treatment with NX-2127 degrades and attenuates BTK and IKZF1/3 levels, as well as inhibits the downstream pro-growth and pro-survival signaling, resulting in loss of viability of MCL cells, including those resistant to covalent BTKi treatment. These findings also show promising anti-MCL activity of NX-2127-based combination with BCL2 or BET inhibitor which merits further in vivo validation.