The ClpP activator ONC‐212 (TR‐31) inhibits BCL2 and B‐cell receptor signaling in CLL

Abstract Despite advances in therapy, a significant proportion of patients with chronic lymphocytic leukemia (CLL) relapse with drug resistant disease. Novel treatment approaches are required, particularly for high risk disease. The imipridones represent a new class of cancer therapy that has been investigated in pre‐clinical and clinical trials against a range of different cancers. We investigated the effects of the imipridone, ONC‐212, against CLL cells cultured under conditions that mimic aspects of the tumour microenvironment and a TP53ko CLL cell line (OSU‐CLL‐TP53ko). ONC‐212 induced dose‐dependent apoptosis, cell cycle arrest and reduced the migration of CLL cells in vitro, including cells from patients with TP53 lesions and OSU‐CLL‐TP53ko cells. The effects of ONC‐212 were associated with protein changes consistent with activation of the mitochondrial protease, CIpP, and the integrated stress response. We also observed inhibition of pathways downstream of the B‐cell receptor (BCR) (AKT and MAPK‐ERK1/2) and a pro‐apoptotic shift in the balance of proteins of the BCL2 family of proteins (BCL2, MCL1, BCLxL, BAX and NOXA). In conclusion, the study suggests ONC‐212 may represent an effective treatment for high risk CLL disease by inhibiting multiple facets of the BCR signaling pathway and the pro‐survival effects of the BCL2‐family proteins.

drug resistance among patients treated with these novel agents is common. Identification of novel treatment approaches remains an important focus of research in CLL.
The imipridones are a novel class of anti-cancer compounds that have shown activity against a variety of solid and haematological malignancies [1][2][3][4]. Pre-clinical studies of ONC-201 [5] have led to several on-going clinical trials in a range of solid tumours, and leukemias [6][7][8]. ONC-212 (TR-31) is a next-generation imipridone that has been reported to have more potent anti-cancer properties than the related compound, ONC-201 [9,10]. The mechanisms of action of ONC-212 include activation of the mitochondrial caseinolytic protease (CIpP) [11,12]. ClpP plays a critical role in mitochondrial protein homeostasis and is commonly over-expressed in malignant cells, including in haematological malignancies [13]. Studies show that both inhibition and activation of ClpP activity may represent therapeutic strategies for this disease [13,12].
There is also evidence that select imipridones also block cell signaling pathways associated with specific G-protein coupled receptors [10] and inhibit components of the unfolded protein response (UPR) [9]. The proliferation of tumour cells often occurs under conditions that induce stress on the endoplasmic reticulum (ER) and activate the UPR [14][15][16].
Under normal homeostatic conditions, the ER chaperone, glucoseregulated protein 78 (Grp78), binds to and decreases activity of the 'stress-sensors' IRE-1, PERK and ATF-6. However, under conditions that induce ER stress, Grp78 dissociates from these sensors and initiates the degradation of misfolded proteins to mitigate cellular damage.
During periods of prolonged ER stress, these adaptive responses can also initiate apoptosis [17,18].
There is strong evidence to suggest that the UPR also plays an important role in the survival and proliferation of CLL cells [19]. ER stress is known to induce BAX/BAK mediated apoptosis [20,21]. This is mediated, at least in part, through activation of ATF4 in the protein kinase R-like ER kinase (PERK) arm of the UPR, which induces expression of the transcription factor CHOP [22,23]. CHOP expression is crucial for ER stress-induced apoptosis [23,24], through transcriptional regulation of the pro-apoptotic members BCL2 family of proteins [25,26]. As BCL2 is overexpressed in CLL cells and plays a significant role in their survival, it follows that the UPR and drugs that decrease components of the UPR warrant further investigation in CLL.
In the current study we demonstrate that the ClpP activator, ONC-212, has significant cytotoxic, cytostatic effects and inhibits the migratory capacity of CLL cells. The data presented suggest that ONC-212 may represent an effective treatment that warrants further investigation in CLL, particularly for patients with high risk disease.

Patient samples
Peripheral blood samples were collected from CLL patients managed at the Royal North Shore Hospital, following informed con-sent. All patients were diagnosed with CLL according to the international workshop on CLL guidelines [27]. Patient samples are detailed in

Generation of the OSU-CLL-TP53ko line
The TP53 knock-out OSU-CLL (OSU-CLL-TP53ko) cell line was generated using a doxycycline-inducible lentiviral CRISPR-Cas9 technique developed at the Walter and Eliza Hall Institute, Parkville, Victoria, Australia [32]. Knock-out of the TP53 gene and the absence of TP53 protein expression were confirmed by direct sequencing and immunoblotting, respectively.

Immunoblotting
Immunoblotting was performed on patient samples and both OSU-CLL

Statistical analyses
All statistical analyses were performed using the T-test function of GraphPad Prism software.

ONC-212 is cytotoxic towards CLL cells in medium alone or in co-culture with CD40L-fibroblasts
ONC-212 was cytotoxic in a dose-dependent manner towards primary CLL cells (n = 10) cultured in medium alone or with CD40L-fibroblasts ( Figure 1A). However, we observed a significant (P < .001) difference in the IC 50 for ONC-212 under the two culture conditions; the IC 50 values were 33.7 +/-11.9 nM and 404 +/-70.6 nM against cells in medium or with CD40L-fibroblasts, respectively. Importantly, we observed no difference in the sensitivity of CLL cells from patients with TP53 lesions (n = 4) to ONC-212.

ONC-212 is cytotoxic against OSU-CLL and OSU-CLL-TP53ko cells
To explore the effects of ONC-212 against TP53 deficient CLL cells, we generated an OSU-CLL cell line in which TP53 was deleted using the CRISPR-Cas9 technology. ONC-212 was cytotoxic against OSU-CLL and OSU-CLL-TP53ko cells in a dose dependent manner

The cytotoxic effects of ONC-212 are consistent with inhibition of BCR signaling and a pro-apoptotic shift in the balance of BCL2 family proteins
The effects of ONC-212 on signaling downstream of the BCR and on expression of BCL-2 family proteins were explored by immunoblotting. Co-culture of primary CLL cells with CD40L-fibroblasts increased expression of total (two/three samples) and phosphorylated AKT and ERK1/2 protein (three/three samples) ( Figure 3B

DISCUSSION
Clinical trials of ibrutinib, idelalisib and venetoclax have revolutionised the treatment of CLL. Despite high response rates, a significant proportion of patients treated with these targeted agents, relapse or develop an aggressive transformation of their disease. While loss or mutation of TP53 remains an indication of poor response and survival rates [33], recent studies have shown that mutations in genes, including BTK, PLC-γ2 and BCL2, are frequently acquired during treatment and are associated with drug resistance [34]. This highlights the need for continued research into novel treatment approaches, particularly for patients with high risk or relapsed disease.
In the current study we demonstrate that the imipridone, ONC-212, has significant activity against primary CLL cells under in vitro conditions that mimic the TME and against a TP53-deficient CLL cell line. However, as observed with other drugs, including fludarabine [35,36] and venetoclax [37], co-culture of primary CLL cells with stromal cells and TP53ko were associated with a significant reduction in the sensitivity of the leukemic cells to ONC-212 ( Figure 3). Although this suggests that higher doses of ONC-212 would be required to overcome the effects of the TME, the IC50 for ONC-212 against healthy

F I G U R E 3
The cytotoxicity of ONC-212 towards CLL cells in co-culture with stromal cells is associated with changes in expression of proteins involved in the UPR and signaling downstream of the B-cell receptor. CLL cells were cultured in medium alone (Medium) or in contact with CD40L-fibroblasts (CD40L). CD40L-fibroblast co-cultured cells were treated with the indicated doses of ONC-212 for 24 hour. The CLL # above each series of immunoblots indicates the individual patient sample analysed, which corresponds to the patients detailed in Table 1 This is the first study to suggest that the mitochondrial protease, CIpP, plays a role in the response of CLL cells to cytotoxic drug treatment. Recent studies demonstrate that activation of CIpP is the principal mechanism of action of ONC-201 and ONC-212, against a range of different cancers [11]. Interestingly, our observation that CIpP expression was lower in untreated TP53ko than WT OSU-CLL cells, suggests that CIpP may be regulated in a TP53-dependent manner and may contribute to the reduced sensitivity of TP53ko cells to ONC-212.
ONC-212 also had effects on the expression of ATF4, Grp78, CIpP and TRAIL in CLL cells ( Figure 3A and Figure S3A) also has significant cytostatic effects on CLL cells (Figures 2A and 2B).
Accumulation of cells in G0/G1 was concomitant with increased ATF4 expression, which is consistent with the role of ATF4 in regulating p21 expression [50].
The current study may appear to contradict that of [19] who showed that BCR signaling was associated with activation of the UPR [19].
However, it is apparent that UPR activation may induce either survival or apoptosis, depending on the cell type and context [51,52].
Despite other studies showing that inhibitors of the UPR increase TRAIL-mediated cell death [53], overall we observed a relatively minor decrease in TRAIL expression in three of the four CLL patient samples following treatment with ONC-212 ( Figure 3A). While CLL cells are reported to be inherently resistant to TRAIL-mediated cell death [54], studies also suggest that CLL cells can be sensitized to TRAILinduced apoptosis under certain conditions [55]. Although no marked changes in TRAIL expression were observed, further studies examining the functionality of this pathway could be conducted to determine whether the drug modulates the sensitivity of CLL cells to TRAILmediated cell death.
The glucose-regulated family of genes (Grp), which include Grp78, acts as chaperones at the ER and is upregulated in response to accumulation of misfolded proteins [56]. The increase in Grp78 expression observed in primary CLL cells co-cultured with stromal cells ( Figure 3A) supports the notion that Grp78 is involved in the adaption of CLL cells to conditions that support their survival. As these in vitro conditions mimic aspects of the lymph node microenvironment, it is conceivable that CLL cells within the lymph nodes may also express high levels of Grp78 as part of their adaption to conditions that drive rapid proliferation. The importance of Grp78 has been identified in different forms of leukemia, including CLL [57,58]. In a mouse model, Grp78 knock-out suppressed the activation of the AKT/mTOR pathway and the accumulation of leukemic blasts [59], suggesting that Grp78 may play an important role in leukemia progression. In other cancers, Grp78 knock-down has also been shown to enhance apoptosis via a mechanism that involves reduced AKT activity [60]. It is also conceivable that the crosstalk between Grp78 and AKT signaling demonstrated in the study by Wey et al, may explain the decreased phosphorylation of AKT we observed with ONC-212 ( Figures 4A and 4B).
In conclusion, the data presented in the current study suggest that ONC-212 may represent a novel therapeutic option for patients with CLL. The efficacy of ONC-212 under conditions that mimic the TME and against TP53 dysfunctional CLL cells raises the possibility that this treatment approach may be particularly effective in patients with a poor prognosis or for patients who develop disease resistant to BCR or BCL2-targeted therapies.

Edwin J. Iwanowicz and Henk Lang have financial interests in Madera
Therapeutics, LLC. In addition, Donald S. Karanewsky is a consultant for Madera Therapeutics, LLC.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.