Jennifer Richmond

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.

Venetoclax responses of pediatric ALL xenografts reveal sensitivity of MLL-rearranged leukemia

The clinical success of the BCL-2-selective BH3-mimetic venetoclax in patients with poor prognosis chronic lymphocytic leukemia (CLL) highlights the potential of targeting the BCL-2-regulated apoptotic pathway in previously untreatable lymphoid malignancies. By selectively inhibiting BCL-2, venetoclax circumvents the dose-limiting, BCL-XL-mediated thrombocytopenia of its less selective predecessor navitoclax, while enhancing efficacy in CLL. We have previously reported the potent sensitivity of many high-risk childhood acute lymphoblastic leukemia (ALL) xenografts to navitoclax. Given the superior tolerability of venetoclax, here we have investigated its efficacy in childhood ALL. We demonstrate that in contrast to the clear dependence of CLL on BCL-2 alone, effective antileukemic activity in the majority of ALL xenografts requires concurrent inhibition of both BCL-2 and BCL-XL We identify BCL-XL expression as a key predictor of poor response to venetoclax and demonstrate that concurrent inhibition of both BCL-2 and BCL-XL results in synergistic killing in the majority of ALL xenografts. A notable exception is mixed lineage leukemia-rearranged infant ALL, where venetoclax largely recapitulates the activity of navitoclax, identifying this subgroup of patients as potential candidates for clinical trials of venetoclax in childhood ALL. Conversely, our findings provide a clear basis for progressing navitoclax into trials ahead of venetoclax in other subgroups.

AKR1C3 is a biomarker of sensitivity to PR-104 in preclinical models of T-cell acute lymphoblastic leukemia.

PR-104, a phosphate ester of the nitrogen mustard prodrug PR-104A, has shown evidence of efficacy in adult leukemia clinical trials. Originally designed to target hypoxic cells, PR-104A is independently activated by aldo-keto-reductase 1C3 (AKR1C3). The aim of this study was to test whether AKR1C3 is a predictive biomarker of in vivo PR-104 sensitivity. In a panel of 7 patient-derived pediatric acute lymphoblastic leukemia (ALL) xenografts, PR-104 showed significantly greater efficacy against T-lineage ALL (T-ALL) than B-cell-precursor ALL (BCP-ALL) xenografts. Single-agent PR-104 was more efficacious against T-ALL xenografts compared with a combination regimen of vincristine, dexamethasone, and l-asparaginase. Expression of AKR1C3 was significantly higher in T-ALL xenografts compared with BCP-ALL, and correlated with PR-104/PR-104A sensitivity in vivo and in vitro. Overexpression of AKR1C3 in a resistant BCP-ALL xenograft resulted in dramatic sensitization to PR-104 in vivo. Testing leukemic blasts from 11 patients confirmed that T-ALL cells were more sensitive than BCP-ALL to PR-104A in vitro, and that sensitivity correlated with AKR1C3 expression. Collectively, these results indicate that PR-104 shows promise as a novel therapy for relapsed/refractory T-ALL, and that AKR1C3 expression could be used as a biomarker to select patients most likely to benefit from such treatment in prospective clinical trials.

A review of new agents evaluated against pediatric acute lymphoblastic leukemia by the Pediatric Preclinical Testing Program.

Acute lymphoblastic leukemia (ALL) in children exemplifies how multi-agent chemotherapy has improved the outcome for patients. Refinements in treatment protocols and improvements in supportive care for this most common pediatric malignancy have led to a cure rate that now approaches 90%. However, certain pediatric ALL subgroups remain relatively intractable to treatment and many patients who relapse face a similarly dismal outcome. Moreover, survivors of pediatric ALL suffer the long-term sequelae of their intensive treatment throughout their lives. Therefore, the development of drugs to treat relapsed/refractory pediatric ALL, as well as those that more specifically target leukemia cells, remains a high priority. As pediatric malignancies represent a minority of the overall cancer burden, it is not surprising that they are generally underrepresented in drug development efforts. The identification of novel therapies relies largely on the reappropriation of drugs developed for adult malignancies. However, despite the large number of experimental agents available, clinical evaluation of novel drugs for pediatric ALL is hindered by limited patient numbers and the availability of effective established drugs. The Pediatric Preclinical Testing Program (PPTP) was established in 2005 to provide a mechanism by which novel therapeutics could be evaluated against xenograft and cell line models of the most common childhood malignancies, including ALL, to prioritize those with the greatest activity for clinical evaluation. In this article, we review the results of >50 novel agents and combinations tested against the PPTP ALL xenografts, highlighting comparisons between PPTP results and clinical data where possible.

Effective Targeting of the P53/MDM2 Axis in Preclinical Models of Infant MLL-Rearranged Acute Lymphoblastic Leukemia

Purpose: Although the overall cure rate for pediatric acute lymphoblastic leukemia (ALL) approaches 90%, infants with ALL harboring translocations in the mixed-lineage leukemia (MLL) oncogene (infant MLL-ALL) experience shorter remission duration and lower survival rates (∼50%). Mutations in the p53 tumor-suppressor gene are uncommon in infant MLL-ALL, and drugs that release p53 from inhibitory mechanisms may be beneficial. The purpose of this study was to assess the efficacy of the orally available nutlin, RG7112, against patient-derived MLL-ALL xenografts. Experimental Design: Eight MLL-ALL patient-derived xenografts were established in immune-deficient mice, and their molecular features compared with B-lineage ALL and T-ALL xenografts. The sensitivity of MLL-ALL xenografts to RG7112 was assessed in vitro and in vivo, and the ability of RG7112 to induce p53, cell-cycle arrest, and apoptosis in vivo was evaluated. Results: Gene-expression analysis revealed that MLL-ALL, B-lineage ALL, and T-ALL xenografts clustered according to subtype. Moreover, genes previously reported to be overexpressed in MLL-ALL, including MEIS1, CCNA1, and members of the HOXA family, were significantly upregulated in MLL-ALL xenografts, confirming their ability to recapitulate the clinical disease. Exposure of MLL-ALL xenografts to RG7112 in vivo caused p53 upregulation, cell-cycle arrest, and apoptosis. RG7112 as a single agent induced significant regressions in infant MLL-ALL xenografts. Therapeutic enhancement was observed when RG7112 was assessed using combination treatment with an induction-type regimen (vincristine/dexamethasone/L-asparaginase) against an MLL-ALL xenograft. Conclusions: The utility of targeting the p53–MDM2 axis in combination with established drugs for the management of infant MLL-ALL warrants further investigation. Clin Cancer Res; 21(6); 1395–405. ©2015 AACR.

Synergism of FAK and tyrosine kinase inhibition in Ph+ B-ALL

BCR-ABL1+ B progenitor acute lymphoblastic leukemia (Ph+ B-ALL) is an aggressive disease that frequently responds poorly to currently available therapies. Alterations in IKZF1, which encodes the lymphoid transcription factor Ikaros, are present in over 80% of Ph+ ALL and are associated with a stem cell-like phenotype, aberrant adhesion molecule expression and signaling, leukemic cell adhesion to the bone marrow stem cell niche, and poor outcome. Here, we show that FAK1 is upregulated in Ph+ B-ALL with further overexpression in IKZF1-altered cells and that the FAK inhibitor VS-4718 potently inhibits aberrant FAK signaling and leukemic cell adhesion, potentiating responsiveness to tyrosine kinase inhibitors, inducing cure in vivo. Thus, targeting FAK with VS-4718 is an attractive approach to overcome the deleterious effects of FAK overexpression in Ph+ B-ALL, particularly in abrogating the adhesive phenotype induced by Ikaros alterations, and warrants evaluation in clinical trials for Ph+ B-ALL, regardless of IKZF1 status.

Acute Sensitivity of Ph-like Acute Lymphoblastic Leukemia to the SMAC-Mimetic Birinapant.

Ph-like acute lymphoblastic leukemia (ALL) is a genetically defined high-risk ALL subtype with a generally poor prognosis. In this study, we evaluated the efficacy of birinapant, a small-molecule mimetic of the apoptotic regulator SMAC, against a diverse set of ALL subtypes. Birinapant exhibited potent and selective cytotoxicity against B-cell precursor ALL (BCP-ALL) cells that were cultured ex vivo or in vivo as patient-derived tumor xenografts (PDX). Cytotoxicity was consistently most acute in Ph-like BCP-ALL. Unbiased gene expression analysis of BCP-ALL PDX specimens identified a 68-gene signature associated with birinapant sensitivity, including an enrichment for genes involved in inflammatory response, hematopoiesis, and cell death pathways. All Ph-like PDXs analyzed clustered within this 68-gene classifier. Mechanistically, birinapant sensitivity was associated with expression of TNF receptor TNFR1 and was abrogated by interfering with the TNFα/TNFR1 interaction. In combination therapy, birinapant enhanced the in vivo efficacy of an induction-type regimen of vincristine, dexamethasone, and L-asparaginase against Ph-like ALL xenografts, offering a preclinical rationale to further evaluate this SMAC mimetic for BCP-ALL treatment. Cancer Res; 76(15); 4579-91. ©2016 AACR.

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.

Xenograft-directed personalized therapy for a patient with post-transplant relapse of ALL

The outcome for ALL relapsing after BM transplantation (BMT) remains poor. International Bone Marrow Transplant Register data suggest that about 30% of patients can be cured with a second BMT and that younger age (o20 years), longer interval between transplants (46 months) and deeper molecular remission contribute to greater likelihood of success. The selection of chemotherapy to achieve deep remission before second BMT is often complicated by the patient’s previous exposures to agents with cumulative toxicities such as anthracyclines and/or slow minimal residual disease (MRD) responses to previous induction therapy. The use of novel therapies targeted to specific ALL biology is attractive and has been pioneered for tyrosine kinase inhibitors in Ph-like ALL. Although integrated genomic analysis may identify novel treatment options for relapsed ALL, it is important to establish if targeted therapy will be more effective than conventional remission induction which works in 60–70% of cases. Xenograft models accurately reproduce ALL in preclinical studies and patient-derived xenografts (PDX) could be used to assess this and to prioritize therapy for individual ALL relapse cases. We demonstrate that biologic, genetic and expression analyses combined with the treatment of a patient’s ALL in PDX mice were able to provide relevant information in time to guide therapy selection for a patient with a post-transplant relapse resulting in a complete molecular remission and enabling a second BMT. A 3-year-old girl diagnosed with pre-B-ALL was treated as a high standard-risk patient following the Children’s Oncology Group protocol (ALL0331) due to a slow early marrow response with high minimal residual disease (MRD410 ; Supplementary Table S1). She completed therapy but had an isolated BM relapse 35 months after diagnosis. The patient was enrolled in the UKALL-R3 trial in the intermediate risk group and had high MRD (1 × 10 ) at end of induction. As per trial recommendation, she received a matched-sibling-donor BMT. She had low level MRD (positive o5 × 10 ) pre-BMT and a second isolated BM relapse occurred 15 months after the first transplant. The family wished to pursue curative therapy, with the plan to induce a third remission followed by a second BMT. Treatment with fludarabine, cytarabine and mitoxantrone (FLAG-M) was commenced 5 days after the second relapse (Figure 1, Table 1). A multi-disciplinary panel was convened to determine whether alternate treatment(s) could be identified and validated in real-time in the event of failure of FLAG-M to achieve a third remission. The panel reviewed diagnostic information, results from the literature and the Pediatric Preclinical Testing Program (PPTP) and chose targeted genetic analyses to be performed to facilitate rapid analysis and enable PDX validation. In particular we tested for a BCR-ABL1-like expression signature including specific targetable fusions, performed phospho-flow cytometry for targetable kinase activity; low-density genomic analysis by multiplex ligation-dependent PCR (MLPA); and screened for activating RAS or JAK mutations. Twenty non-irradiated, immune-deficient, NOD/SCID/IL-2 receptor gamma /− (NOD.Cg-Prkdc Il2rg/SzJ, NSG) mice were inoculated by tail vein injection 8 days after the 2nd relapse with 2 × 10 BM cells (from the 2nd relapse) to establish the PDX.