BCL-2 inhibition – stemming the tide of myeloid malignancies

Abstract

Leukemia stem cells (LSC) are likely to be the source for both therapeutic resistance and relapse in myeloid malignancies. (Lagadinou et al., 2013) and (Goff et al., 2013) find that LSC can be selectively targeted by small molecule antagonists of anti-apoptotic BCL-2 family proteins.

Cancer stem cells possess the capacity to self-renew as well as generate the heterogeneous cancer cells that comprise a tumor. Although the theory of cancer stem cells was proposed nearly 50 years ago, prospectively identifying cancer stem cells remains a challenge, as does killing these quiescent, chemoresistant cancer stem cells. Myeloid cancers, particularly chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) have been useful model systems in the establishment of cancer stem cells (Wang & Dick, 2005). In this week's Cell Stem Cell, Lagadinou et al., 2013 and Goff et al., 2013 find that small molecule inhibition of anti-apoptotic Bcl-2 family members selectively harms leukemia cells that possess stem cell properties.

Instead of using canonical cell surface markers to identify leukemia stem cells (LSCs), Lagadinou et al. apply a functional approach. They hypothesize that LSCs, being quiescent in nature, have lower metabolic activity and thus lower reactive oxygen species (ROS) production. The authors profile primary acute myeloid leukemia cells (AML) for their ROS levels and find that ROS-low cells not only exhibit less proliferation and a higher fraction of cells in G₀, but also exhibit an increased ability to engraft in immunodeficient mice compared to ROS-high cells. In order to identify a mechanism underlying this phenomenon, they perform RNA-seq on ROS-low and ROS-high cells and find that the anti-apoptotic BH3-only family member BCL-2 is up-regulated in ROS-low cells. This finding is validated by qRT-PCR and immunoblot analysis. Importantly, Lagadinou et al. demonstrate that inhibiting BCL-2 with the battle-tested BCL-2 family inhibitor ABT-263 (Roberts et al., 2012) selectively kills ROS-low cells in vivo. In addition, ex vivo treatment of AML cells with ABT-263 inhibits engraftment of AML cells into NSG mice.

The authors' exploitation of ROS measurements to functionally identify LSCs and BCL-2 dependent subsets is both novel and potentially of great utility. The perturbation of metabolism and oxygen consumption by BCL-2 inhibition may require further clarification, however. It is known that in sensitive cells, BCL-2 inhibition can cause mitochondrial outer membrane permeabilization (MOMP) very rapidly, even within an hour (Del Gaizo Moore et al., 2008; Vogler et al., 2008). This permeabilization by itself could cause significant perturbations in oxidative phosphorylation and oxygen consumption. Thus, it needs to be clarified whether the metabolic effects of BCL-2 inhibition observed by the authors occur before or after MOMP.

Similar to Lagadinou et al, Goff et al., find that inhibition of BCL-2 (and its family members) selectively target LSCs in primary human CML cells. Instead of taking a functional approach, the authors more traditionally define CML LSCs as CD34⁺CD⁺38 Lin⁻. They find that isolated human BC LSCs transplanted into RAG^−/−γ^−/− which engraft into the bone marrow are more quiescent and chemoresistant compared to those that engraft at the tumor site, spleen or liver. Based on a fairly modest relative increase in expression of anti-apoptotic proteins like BCL-2 in blast crisis (BC) LSCs compared to normal or chronic phase (CP) CD34⁺CD⁺38Lin⁻ cells, the authors treat the cells with sabutoclax, developed as a pan-BCL-2 inhibitor. They observe that BC progenitor cells are more sensitive to sabutoclax compared to normal progenitor cells. Further, combination therapy of dasatinab and sabutoclax of CML cells serially transplanted into mice cause a modest increase in life span compared to untreated or single agent-treated animals (median survival increased from ∼75 days to ∼85 days).

To clarify that the observations are truly a leukemia stem cell based effect of direct inhibition of BCL-2 family proteins, a couple of issues may require future clarification. First, it is not completely clear that the mechanism of in vivo toxicity of sabutoclax is solely due to inhibition of BCL-2 family proteins. This is pertinent because gossypol, from which sabutoclax was derived, exhibits mechanisms of killing that are distinct from direct inhibition of BCL-2 family proteins (Van Delft et al., 2006). In this respect, it will be interesting to see whether at effective doses sabutoclax induces thrombocytopenia in mammals in vivo. While thrombocytopenia can be a problem clinically, it is a useful in vivo biomarker of BCL-XL inhibition, as circulating platelets have been found to be dependent on BCL-XL for survival (Mason et al., 2007). In addition, it is not clear whether these results from BC CML cells can be extended to stem cells from de novo AML. In the former case, the authors identified their stem cells as bearing a CD34⁺/CD38⁺ phenotype, while in the latter case, the CD34⁺/CD38⁻ population is more commonly thought to bear the stem cells (Wang & Dick, 2005).

An exciting, translational message that emerges from both papers is that inhibition of BCL-2 is a promising approach to the treatment of myeloid malignancies. These results are consistent with prior studies that found that bulk and stem-like AML cells are more dependent on BCL-2 than normal HSC, and are therefore more sensitive to BCL-2 inhibition by small molecules (Konopleva et al., 2006; Vo et al., 2012). This evidence comes at a particularly propitious time with the recent introduction of ABT-199 into clinical trials (Souers et al., 2013). As discussed briefly above, the dose-limiting toxicity of ABT-263 thrombocytopenia, caused by the inhibition of BCL-XL, is a particular problem in the treatment of AML patients that usually already suffer from thrombocytopenia. ABT-199, by selectively targeting BCL-2 but not BCL-XL, avoids this toxicity, likely making its use in AML better tolerated. With the identification of a therapeutic index and elimination of a major toxicity, it would seem that conditions are ripe for clinical testing of BCL-2 inhibition in myeloid cancers.