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C. Ciardullo et al.
response to MDM2 antagonists and other biomarkers should be sought. In fact, in addition to p53 dysfunction resulting from TP53 mutations and/or deletions, human cancers may display p53 suppression as a consequence of upregulation of MDM2 expression.47 MDM2, which can enhance tumorigenic potential and resistance to apopto- sis, has also been reported to be overexpressed in 50-70% of CLL cases;43,44 it is, therefore, reasonable to hypothesize that aberrant expression of MDM2 could be an indicator of response to MDM2 inhibitors. However, in our study the basal mRNA expression of MDM2 was not signifi- cantly different between RG7388-sensitive samples (LC50 <1 μM) and more resistant CLL samples (LC50 >1 μM). Moreover, we found no significant correlation between basal MDM2 expression or MDM2 fold-induction and LC50 values, supporting previous observations that MDM2 overexpression does not have an impact on functional activation of p53 or MDM2 inhibitor-induced cytotoxicity in CLL.45,46 In contrast, a recent study showed that MDM2 protein expression in blasts may identify patients with acute myeloid leukemia likely to exhibit better outcomes to RG7388-based therapy.33 Quantification of MDM2 basal levels might, therefore, also be clinically relevant in other hematologic malignancies in order to predict sensi- tivity to MDM2 inhibitors.
The main concern regarding p53-reactivating therapies is their effect on normal cells. The activation of functional p53 by MDM2 inhibitors could elicit different cellular responses in tumor cells compared to normal cells. However, there is a paucity of data on the effect of new- generation MDM2 antagonists on normal cells, especially CD34+ hematopoietic stem cells in which drug-induced cytotoxicity can result in the dose-limiting cytopenia that has been reported in early clinical trials of these agents. Although some initial studies (using Nutlin-3 and MI-219) suggested that MDM2 inhibition results in different cellu- lar responses in normal and tumor cells,38-41 the pattern of p53-dependent gene expression induced by MDM2 inhi- bition in primary CLL cells versus normal blood cells has not been investigated.
Here, we show for the first time that the expression of p53-target genes in response to RG7388 in normal periph- eral blood and bone marrow cells (including positively- selected CD34+ hematopoietic progenitors) is distinct from that in primary CLL cells. Induction of the pro-apop- totic PUMA gene after RG7388 treatment was the domi- nant response in CLL cells. This contrasted with the response of normal blood cells and CD34+ hematopoietic stem cells, in which activation of apoptosis was weak or absent and upregulation of the negative feedback regula- tor MDM2 dominated over that of pro-apoptotic target genes. Interestingly, the induction of CDKN1A was also higher in normal PBMC than in p53-functional CLL cells, suggesting that reactivation of p53 in normal, circulating blood cells by MDM2 inhibitors fails to elicit the predom- inant cell-death signal seen in CLL cells. In CD34+ cells, gene expression and cell cycle distribution changes also suggest that cell-cycle arrest and an effective re-establish- ment of the MDM2-p53 negative feedback loop, rather than apoptosis, might be the main effects elicited by RG7388. These findings provide a mechanistic rationale for observations on the use of first-generation MDM2 antagonists that have suggested a predominant, reversible growth arrest as a primary response of normal cells to MDM2 inhibition.38-41 Consistent with this, activation of
caspase 3/7 and cytotoxicity upon exposure to RG7388 were significantly less in normal blood and bone marrow cells than in primary CLL cells.
Although p53 is activated by MDM2 inhibitors in both normal and tumor cells with functional p53, the gene expression signature and the cytotoxic effect induced by p53 activation in these two settings are markedly distinct, which translates into different cell fates and provides a therapeutic index with significant implications for the potential applications of MDM2 inhibitors as new anti- cancer agents. Of additional importance, RG7388 also effectively blocked proliferation signals provided external- ly to CLL cells in vitro to model the microenvironment (CD40L and IL4), which are crucial in vivo stimuli for pro- liferation of leukemic cells in lymph nodes and bone mar- row.
IgM stimulation of BCR signaling has been reported to increase protein levels of MCL1, but not BCL2, and to pro- mote the survival of CLL cells.48 Because of the importance of BCR signaling in CLL it would be interesting to explore the effect of IgM and/or IL4 stimulation on the response of CLL cells to MDM2 inhibitors, with and without spe- cific inhibitors of BCL2 and MCL1. IgM stimulation of BCR signaling would also provide a potential ex vivo model simulating the lymph node microenvironment for investigation of combination treatments with ibrutinib.
We cannot rule out that conformational changes in BAX may be important, although BAX expression changed little compared to the clear large changes in PUMA expression. A transcription-independent role of p53 in CLL cell apop- tosis, involving direct interactions of p53 with mitochon- drial anti-apoptotic proteins such as BCL2, has been sug- gested.42 We favor a model in which p53 transcription- dependent and -independent mechanisms work hand in hand. Stabilization of p53 and upregulation of p53 tran- scriptional target genes, including predominantly pro- apoptotic genes, particularly PUMA, are the earliest and necessary events in the response of CLL cells to MDM2- p53 binding interaction inhibitors. Gene knockout mouse studies show that PUMA is necessary for apoptosis and p53 induction on its own is not sufficient. Studies on BAX nullizygous mice concluded that PUMA provides the crit- ical link between p53 and BAX and is both necessary and sufficient to mediate DNA damage-induced apoptosis.49 Furthermore PUMA knockout studies in mice show reca- pitulation of virtually all apoptotic deficiency in p53 knockout mice.50 It is therefore reasonable to link the major induction of PUMA by MDM2 inhibitor treatment of CLL cells with an important role in their sensitivity to the induction of apoptosis by these compounds. The absence of any marked downregulation of BCL family anti-apoptotic gene expression in our current study ruled out suppression of the transcriptional expression of these genes as a major contributory mechanism to the response to MDM2 inhibitors.
In considering the therapeutic potential of MDM2 inhibitors in CLL, it should also be emphasized that, despite improvements in patients’ response rate using chemo-immunotherapy combinations or BCR-antago- nists, none of the current therapeutic regimens is cura- tive.8,9 They are subject to limitations, including the evolu- tion of drug resistance mechanisms. Resistance as a result of mutations in the venetoclax-binding domain of BCL2 has been reported in a high proportion of patients who relapse after treatment with venetoclax.51 Similarly, a high
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haematologica | 2019; 104(12)