Jessica M. Salmon

Adult Hematopoietic Stem and Progenitor Cells Require Either Lyl1 or Scl for Survival

Scl and Lyl1 encode two related basic-helix-loop-helix transcription factors implicated in T cell acute lymphoblastic leukemia. Previous studies showed that Scl is essential for embryonic and adult erythropoiesis, while Lyl1 is important for B cell development. Single-knockout mice have not revealed an essential function for Scl or Lyl1 in adult hematopoietic stem cells (HSCs). To determine if maintenance of HSCs in single-knockout mice is due to functional redundancy, we generated Lyl1;Scl-conditional double-knockout mice. Here, we report a striking genetic interaction between the two genes, with a clear dose dependence for the presence of Scl or Lyl1 alleles for HSC function. Bone marrow repopulation assays and analyses demonstrated rapid loss of hematopoietic progenitors due to apoptosis. The function of HSCs could be rescued by a single allele of Lyl1 but not Scl. These results show that expression of at least one of these factors is essential for maintenance of adult HSC function.

Differentiation therapy for the treatment of t(8;21) acute myeloid leukemia using histone deacetylase inhibitors

Epigenetic modifying enzymes such as histone deacetylases (HDACs), p300, and PRMT1 are recruited by AML1/ETO, the pathogenic protein for t(8;21) acute myeloid leukemia (AML), providing a strong molecular rationale for targeting these enzymes to treat this disease. Although early phase clinical assessment indicated that treatment with HDAC inhibitors (HDACis) may be effective in t(8;21) AML patients, rigorous preclinical studies to identify the molecular and biological events that may determine therapeutic responses have not been performed. Using an AML mouse model driven by expression of AML1/ETO9a (A/E9a), we demonstrated that treatment of mice bearing t(8;21) AML with the HDACi panobinostat caused a robust antileukemic response that did not require functional p53 nor activation of conventional apoptotic pathways. Panobinostat triggered terminal myeloid differentiation via proteasomal degradation of A/E9a. Importantly, conditional A/E9a deletion phenocopied the effects of panobinostat and other HDACis, indicating that destabilization of A/E9a is critical for the antileukemic activity of these agents.

Functional but Abnormal Adult Erythropoiesis in the Absence of the Stem Cell Leukemia Gene

ABSTRACT Previous studies have indicated that the stem cell leukemia gene (SCL) is essential for both embryonic and adult erythropoiesis. We have examined erythropoiesis in conditional SCL knockout mice for at least 6 months after loss of SCL function and report that SCL was important but not essential for the generation of mature red blood cells. Although SCL-deleted mice were mildly anemic with increased splenic erythropoiesis, they responded appropriately to endogenous erythropoietin and hemolytic stress, a measure of late erythroid progenitors. However, SCL was more important for the proliferation of early erythroid progenitors because the predominant defects in SCL-deleted erythropoiesis were loss of in vitro growth of the burst-forming erythroid unit and an in vivo growth defect revealed by transplant assays. With respect to erythroid maturation, SCL-deleted proerythroblasts could generate more mature erythroblasts and circulating red blood cells. However, SCL was required for normal expression of TER119, one of the few proposed target genes of SCL. The unexpected finding that SCL-independent erythropoiesis can proceed in the adult suggests that alternate factors can replace the essential functions of SCL and raises the possibility that similar mechanisms also explain the relatively minor defects previously observed in SCL-null hematopoietic stem cells.

The caspase-8 inhibitor emricasan combines with the SMAC mimetic birinapant to induce necroptosis and treat acute myeloid leukemia

The combination of a SMAC mimetic and a caspase inhibitor kills AML cells by inducing necroptosis. Giving leukemia a SMAC Second mitochondria-derived activator of caspases, or SMAC, is a protein involved in apoptosis, a mechanism of cell death that is commonly targeted by cancer therapies. SMAC mimetics are drugs designed to mimic the action of SMAC. Now, a pair of related articles provides insights into the effects of SMAC mimetics in leukemia. For acute lymphocytic leukemia, McComb et al. show that a SMAC mimetic called birinapant works best when it can activate two different types of cell death: apoptosis and necroptosis. For acute myelocytic leukemia, Brumatti et al. show that birinapant is particularly effective when combined with a caspase inhibitor, which shuts off the apoptotic pathway and promotes cell death by necroptosis. These findings should be helpful for identifying patients most likely to benefit from treatment with SMAC mimetics and selecting effective treatment combinations for these patients. Resistance to chemotherapy is a major problem in cancer treatment, and it is frequently associated with failure of tumor cells to undergo apoptosis. Birinapant, a clinical SMAC mimetic, had been designed to mimic the interaction between inhibitor of apoptosis proteins (IAPs) and SMAC/Diablo, thereby relieving IAP-mediated caspase inhibition and promoting apoptosis of cancer cells. We show that acute myeloid leukemia (AML) cells are sensitive to birinapant-induced death and that the clinical caspase inhibitor emricasan/IDN-6556 augments, rather than prevents, killing by birinapant. Deletion of caspase-8 sensitized AML to birinapant, whereas combined loss of caspase-8 and the necroptosis effector MLKL (mixed lineage kinase domain-like) prevented birinapant/IDN-6556–induced death, showing that inhibition of caspase-8 sensitizes AML cells to birinapant-induced necroptosis. However, loss of MLKL alone did not prevent a caspase-dependent birinapant/IDN-6556–induced death, implying that AML will be less likely to acquire resistance to this drug combination. A therapeutic breakthrough in AML has eluded researchers for decades. Demonstrated antileukemic efficacy and safety of the birinapant/emricasan combination in vivo suggest that induction of necroptosis warrants clinical investigation as a therapeutic opportunity in AML.

Targeting p38 or MK2 Enhances the Anti-Leukemic Activity of Smac-Mimetics.

Birinapant is a smac-mimetic (SM) in clinical trials for treating cancer. SM antagonize inhibitor of apoptosis (IAP) proteins and simultaneously induce tumor necrosis factor (TNF) secretion to render cancers sensitive to TNF-induced killing. To enhance SM efficacy, we screened kinase inhibitors for their ability to increase TNF production of SM-treated cells. We showed that p38 inhibitors increased TNF induced by SM. Unexpectedly, even though p38 is required for Toll-like receptors to induce TNF, loss of p38 or its downstream kinase MK2 increased induction of TNF by SM. Hence, we show that the p38/MK2 axis can inhibit or promote TNF production, depending on the stimulus. Importantly, clinical p38 inhibitors overcame resistance of primary acute myeloid leukemia to birinapant.

The role of p21 waf1/cip1 and p27 Kip1 in HDACi-mediated tumor cell death and cell cycle arrest in the Eμ-myc model of B-cell lymphoma

Following the establishment of histone deacetylases (HDACs) as promising therapeutic targets for the reversal of aberrant epigenetic states associated with cancer, the development of HDAC inhibitors (HDACi) and their underlying mechanisms of action has been a significant area of scientific interest. HDACi induce diverse biological responses including the inhibition of cell proliferation by blocking progression through the G1 or G2/M phases of the cell cycle. As a putative tumor-suppressor protein, p21waf1/cip1 influences cell proliferation by inhibiting the activity of cyclin–cyclin-dependent kinase (CDK) complexes at the G1/S and G2/M cell cycle checkpoints. HDACi transcriptionally activate CDKN1A, and it has been proposed that induction of p21waf1/cip1 can determine if a cell undergoes apoptosis or cell cycle arrest following HDACi treatment. In the Eμ-myc transgenic mouse model of B-cell lymphoma, knockout of cdkn1a had no effect on disease latency, indicating that p21waf1/cip1 did not function as a tumor suppressor in this system. Although HDACi robustly induced expression of p21waf1/cip1 in wild-type Eμ-myc lymphomas, deletion of cdkn1a did not sensitize the lymphoma cells to HDACi-induced apoptosis and HDACi-induced cell cycle arrest still occurred. However, knockdown of cdkn1b in cdkn1a knockout lymphomas resulted in defective vorinostat-mediated arrest at G1/S indicating an essential role of p27Kip1 in mediating this biological response to vorinostat. These data demonstrate that induction of cdkn1a does not regulate HDACi-mediated tumor cell apoptosis and refute the notion that p21waf1/cip1 is an obligate mediator of HDACi-induced cell cycle arrest.

Concise Review: Blood Relatives: Formation and Regulation of Hematopoietic Stem Cells by the Basic Helix-Loop-Helix Transcription Factors Stem Cell Leukemia and Lymphoblastic Leukemia-Derived Sequence 1

The basic helix‐loop‐helix (bHLH) proteins are a large family of transcription factors that regulate the formation and fate of tissue stem cells. In hematopoiesis, the two major bHLH factors are stem cell leukemia (SCL) and lymphoblastic leukemia‐derived sequence 1 (LYL1), both identified more than 20 years ago in chromosomal translocations occurring in T‐cell acute lymphoblastic leukemia. SCL was termed the master regulator of hematopoiesis following the observation that SCL knockout mice die from complete lack of blood formation. However, once established, SCL is no longer required for maintenance of hematopoiesis. Pull‐down experiments together with add‐back experiments in SCL‐null embryonic stem cells and generation of mice carrying a germline DNA binding mutation of SCL demonstrates that most of SCL function is mediated through the formation of a large DNA binding multiprotein complex with both repressor and activator potential. Recent genome‐wide binding studies in a hematopoietic stem progenitor cell line suggest that SCL and LYL1 preferentially bind target DNA sequences as components of a heptad of transcription factors. LYL1, a paralog of SCL has been the forgotten sibling until recent mouse studies demonstrated that LYL1 replaced the function of SCL in adult hematopoiesis. Why LYL1 can replace the function of SCL for the maintenance but not formation of hematopoiesis remains a fundamental question. This review will compare and contrast the roles of these two transcription factors in hematopoiesis focusing on recent functional and genome‐wide binding studies. STEM CELLS2012;30:1053–1058

Correction: Targeting p38 or MK2 Enhances the Anti-Leukemic Activity of Smac-Mimetics (Cancer Cell (2016) 30(3) (499–500) (S1535610816000350) (10.1016/j.ccell.2016.01.006))

Corrections Targeting p38 or MK2 Enhances the Anti-Leukemic Activity of Smac-Mimetics Najoua Lalaoui, Kay Hänggi, Gabriela Brumatti, Diep Chau, Nhu-Y.N. Nguyen, Lazaros Vasilikos, Lisanne M. Spilgies, Denise A. Heckmann, Chunyan Ma, Margherita Ghisi, Jessica M. Salmon, Geoffrey M. Matthews, Elisha de Valle, Donia M. Moujalled, Manoj B. Menon, Sukhdeep Kaur Spall, Stefan P. Glaser, Jennifer Richmond, Richard B. Lock, StephenM. Condon, Raffi Gugasyan, Matthias Gaestel, Mark Guthridge, RickyW. Johnstone, LenkaMunoz, AndrewWei, Paul G. Ekert, David L. Vaux, W. Wei-Lynn Wong, and John Silke* *Correspondence: silke@wehi.edu.au http://dx.doi.org/10.1016/j.ccell.2016.08.009 (Cancer Cell 29, 145–158; February 8, 2016) After the publication of this paper, the authors found four small errors in Figure 3. In Figure 3A, the label p38 on the right side of the second blot from the top should be p38, and the label MK2 on the right side of the second blot from the bottom should be MK2. In addition, the pTAK1 and pMK2 blots in Figure 3C were inadvertently stretched out during figure preparation. These errors have now been corrected here and in the article online. The authors apologize for these errors and any inconvenience that may have resulted.

Combining the differentiating effect of panobinostat with the apoptotic effect of arsenic trioxide leads to significant survival benefit in a model of t(8;21) acute myeloid leukemia

BackgroundOne of the most frequently found abnormalities in acute myeloid leukemia (AML) is the t(8;21)(q22;q22) translocation, which is seen in around 15% of patients. This translocation results in the production of the AML1/ETO (A/E) fusion protein and commonly involves cooperating activating mutations of RAS. AE9a encodes a C-terminally truncated A/E protein of 575 amino acids that retains the ability to recruit histone deacetylases (HDACs). Expression of AE9a leads to rapid development of leukemia in experimental mouse systems. We have recently shown that treatment of mice bearing A/E9a;NrasG12D tumors with the histone deacetylase inhibitor (HDACi) panobinostat leads to degradation of the A/E9a fusion protein, cell cycle arrest, differentiation of AML blasts into mature granulocytes and prolonged survival. Herein, we sought to enhance this therapeutic effect.FindingsCombined treatment of mice bearing A/E9a;NrasG12D leukemias with panobinostat and arsenic trioxide (ATO) resulted in a significant survival advantage compared to mice treated with either agent alone. Moreover, some of the mice treated with the panobinostat/ATO combination showed complete tumor responses and remained in remission for over 220 days. Panobinostat caused differentiation of A/E9a;NrasG12D cells while ATO induced apoptosis of the leukemic cells, an effect that was enhanced following co-treatment with panobinostat.ConclusionsOur results indicate that leukemic blast differentiation mediated by panobinostat combined with induction of apoptosis by ATO could be therapeutically beneficial and should be considered for patients with t(8;21) AML.