The circadian rhythm, defined as daily oscillation in various biological processes, is controlled by an endogenous clock in most normal cells in the body. Prior research has established that hematopoietic stem cells (HSCs) are regulated by a circadian rhythm, which is established by cell-autonomous expression of core circadian clock genes as well as by non-cell autonomous inputs from the nervous system and microenvironment. Given the importance of circadian regulation of cell proliferation, there has been great interest in understanding the molecular links between the circadian clock and growth regulation, especially in how this regulation might go awry in cancer. However, research linking circadian rhythm and cancer has been fraught with inconsistencies, including the description of a potential tumor suppressor role that was later disputed by null findings.
Now, Dr. Rishi V. Puram and colleagues have identified a leukemia-specific requirement for core circadian clock genes. This finding came about via an in vivo RNA interference screen in mouse acute myeloid leukemia (AML) cells driven by MLL-AF9. This model is enriched for leukemia stem cells (LSCs), and the authors generated short hairpin RNA (shRNA) targeting genes encoding 152 DNA-binding proteins that have higher expression in HSCs or LSCs versus myeloid progenitors. Besides confirming the importance of established regulators of LSC function (such as HoxA9 and Meis1), the screen also identified genes encoding the core circadian clock proteins Clock and Bmal1 as among the top-scoring hits. This discovery contrasts previous research that showed that these genes were silenced in leukemia and posits that loss of physiologic circadian rhythm contributes to leukemia development.
Normal and Malignant Hematopoietic Stem Cells Have Circadiandependent Regulation of Gene Expression but Leukemic Stem Cells in AML Require Core Circadian Clock Genes. (A) Circadian rhythm is generated by cyclic expression of BMAL1, which is high at the beginning of a subjective day and low at the beginning of a subjective night. When BMAL1 is expressed it forms a heterodimeric transcription factor with CLOCK to directly promote the expression of CRY, PER, and REV-ERBα. CRY, PER, and REV-ERBα then serve to inhibit the expression of BMAL1 and/ or the heterodimerization of BMAL1 to CLOCK. (B) The cyclic expression of these genes are present in normal as well as leukemic stem cells in vivo and Puram et al. have now found that BMAL1 and CLOCK are 1) upregulated by the MLLAF9 fusion oncoprotein and 2) preferentially required in leukemic stem cells over normal hematopoietic stem cells. Moreover, the malignant cells were preferentially sensitive to the compound SR9011, which is an agonist of REV-ERBα and downregulates BMAL1 expression.
Normal and Malignant Hematopoietic Stem Cells Have Circadiandependent Regulation of Gene Expression but Leukemic Stem Cells in AML Require Core Circadian Clock Genes. (A) Circadian rhythm is generated by cyclic expression of BMAL1, which is high at the beginning of a subjective day and low at the beginning of a subjective night. When BMAL1 is expressed it forms a heterodimeric transcription factor with CLOCK to directly promote the expression of CRY, PER, and REV-ERBα. CRY, PER, and REV-ERBα then serve to inhibit the expression of BMAL1 and/ or the heterodimerization of BMAL1 to CLOCK. (B) The cyclic expression of these genes are present in normal as well as leukemic stem cells in vivo and Puram et al. have now found that BMAL1 and CLOCK are 1) upregulated by the MLLAF9 fusion oncoprotein and 2) preferentially required in leukemic stem cells over normal hematopoietic stem cells. Moreover, the malignant cells were preferentially sensitive to the compound SR9011, which is an agonist of REV-ERBα and downregulates BMAL1 expression.
The molecular mechanisms regulating the circadian clock involves the asynchronous expression of core clock genes CLOCK and BMAL1 with another set of genes (CRY1, CRY2, PER1, PER2, REV-ERB-α, and REV-ERB-β) which reciprocally regulate one another in feedback loops (Figure). In addition to validating the requirement of CLOCK and BMAL1 expression in mouse and human MLL-rearranged AML cells, the authors also evaluated the requirement for these genes in normal hematopoiesis. Despite the fact that both normal and leukemic hematopoiesis have rhythmic, circadian-dependent regulation of gene expression, Bmal1 was not required for normal adult hematopoiesis in mice.
Given the preferential requirement for circadian clock gene expression in malignant relative to normal hematopoiesis, the authors next tested the effect of a small-molecule REV-ERBα agonist compound in MLL-rearranged leukemia. By stimulating REV-ERB activity, this compound reduced BMAL1 expression as well as the expression of other genes required for MLL-rearranged leukemogenesis (Figure). This drug had a less dramatic effect on CD34+ cord blood cells, indicating that there might be a therapeutic window for inhibiting the circadian clock machinery in leukemia while sparing normal HSCs.
In Brief
This work advances the understanding of the role of cell intrinsic circadian rhythm mechanisms in LSCs and normal HSCs. It will now be important to validate the preferential requirement for core circadian clock genes in leukemia not dependent on rearranged MLL as well as primary samples from AML patients with wider genetic backgrounds. Moreover, it will be very interesting to directly compare the circadian rhythms of normal HSCs versus LSCs in vivo. However, the models used in this experiment were robust and these experiments set the stage for further research into this area as a novel potential target for anti-leukemic therapy.
