Targeting refractory/recurrent neuroblastoma and osteosarcoma with anti-CD3×anti-GD2 bispecific antibody armed T cells.

BACKGROUND: The survival benefit observed in children with neuroblastoma (NB) and minimal residual disease who received treatment with anti-GD2 monoclonal antibodies prompted our investigation into the safety and potential clinical benefits of anti-CD3×anti-GD2 bispecific antibody (GD2Bi) armed T cells (GD2BATs). Preclinical studies demonstrated the high cytotoxicity of GD2BATs against GD2+cell lines, leading to the initiation of a phase I/II study in recurrent/refractory patients. METHODS: The 3+3 dose escalation phase I study (NCT02173093) encompassed nine evaluable patients with NB (n=5), osteosarcoma (n=3), and desmoplastic small round cell tumors (n=1). Patients received twice-weekly infusions of GD2BATs at 40, 80, or 160×106 GD2BATs/kg/infusion complemented by daily interleukin-2 (300,000 IU/m2) and twice-weekly granulocyte macrophage colony-stimulating factor (250 µg/m2). The phase II segment focused on patients with NB at the dose 3 level of 160×106 GD2BATs/kg/infusion. RESULTS: Of the 12 patients enrolled, 9 completed therapy in phase I with no dose-limiting toxicities. Mild and manageable cytokine release syndrome occurred in all patients, presenting as grade 2-3 fevers/chills, headaches, and occasional hypotension up to 72 hours after GD2BAT infusions. GD2-antibody-associated pain was minimal. Median overall survival (OS) for phase I and the limited phase II was 18.0 and 31.2 months, respectively, with a combined OS of 21.1 months. A phase I NB patient had a complete bone marrow response with overall stable disease. In phase II, 10 of 12 patients were evaluable: 1 achieved partial response, and 3 showed clinical benefit with prolonged stable disease. Over 50% of evaluable patients exhibited augmented immune responses to GD2+targets post-GD2BATs, as indicated by interferon-gamma (IFN-γ) EliSpots, Th1 cytokines, and/or chemokines. CONCLUSIONS: This study demonstrated the safety of GD2BATs up to 160×106 cells/kg/infusion. Coupled with evidence of post-treatment endogenous immune responses, our findings support further investigation of GD2BATs in larger phase II clinical trials.

Panobinostat sensitizes AraC-resistant AML cells to the combination of azacitidine and venetoclax.

The majority of acute myeloid leukemia (AML) patients respond to intensive induction therapy, consisting of cytarabine (AraC) and an anthracycline, though more than half experience relapse. Relapsed/refractory (R/R) AML patients are difficult to treat, and their clinical outcomes remain dismal. Venetoclax (VEN) in combination with azacitidine (AZA) has provided a promising treatment option for R/R AML, though the overall survival (OS) could be improved (OS ranges from 4.3 to 9.1 months). Overexpression of c-Myc is associated with chemoresistance in AML. Histone deacetylase (HDAC) inhibitors have been shown to suppress c-Myc and enhance the antileukemic activity of VEN, as well as AZA, though combination of all three has not been fully explored. In this study, we investigated the HDAC inhibitor, panobinostat, in combination with VEN + AZA against AraC-resistant AML cells. Panobinostat treatment downregulated c-Myc and Bcl-xL and upregulated Bim, which enhanced the antileukemic activity of VEN + AZA against AraC-resistant AML cells. In addition, panobinostat alone and in combination with VEN + AZA suppressed oxidative phosphorylation and/or glycolysis in AraC-resistant AML cells. These findings support further development of panobinostat in combination with VEN + AZA for the treatment of AraC-resistant AML.

The Imipridone ONC213 Targets α-Ketoglutarate Dehydrogenase to Induce Mitochondrial Stress and Suppress Oxidative Phosphorylation in Acute Myeloid Leukemia.

Eradication of acute myeloid leukemia (AML) is therapeutically challenging; many patients succumb to AML despite initially responding to conventional treatments. Here, we showed that the imipridone ONC213 elicits potent antileukemia activity in a subset of AML cell lines and primary patient samples, particularly in leukemia stem cells, while producing negligible toxicity in normal hematopoietic cells. ONC213 suppressed mitochondrial respiration and elevated α-ketoglutarate by suppressing α-ketoglutarate dehydrogenase (αKGDH) activity. Deletion of OGDH, which encodes αKGDH, suppressed AML fitness and impaired oxidative phosphorylation, highlighting the key role for αKGDH inhibition in ONC213-induced death. ONC213 treatment induced a unique mitochondrial stress response and suppressed de novo protein synthesis in AML cells. Additionally, ONC213 reduced the translation of MCL1, which contributed to ONC213-induced apoptosis. Importantly, a patient-derived xenograft from a relapsed AML patient was sensitive to ONC213 in vivo. Collectively, these findings support further development of ONC213 for treating AML. SIGNIFICANCE: In AML cells, ONC213 suppresses αKGDH, which induces a unique mitochondrial stress response, and reduces MCL1 to decrease oxidative phosphorylation and elicit potent antileukemia activity. See related commentary by Boët and Sarry, p. 950.

Enhancing anti-AML activity of venetoclax by isoflavone ME-344 through suppression of OXPHOS and/or purine biosynthesis in vitro.

Venetoclax (VEN), in combination with low dose cytarabine (AraC) or a hypomethylating agent, is FDA approved to treat acute myeloid leukemia (AML) in patients who are over the age of 75 or cannot tolerate standard chemotherapy. Despite high response rates to these therapies, most patients succumb to the disease due to relapse and/or drug resistance, providing an unmet clinical need for novel therapies to improve AML patient survival. ME-344 is a potent isoflavone with demonstrated inhibitory activity toward oxidative phosphorylation (OXPHOS) and clinical activity in solid tumors. Given that OXPHOS inhibition enhances VEN antileukemic activity against AML, we hypothesized that ME-344 could enhance the anti-AML activity of VEN. Here we report that ME-344 enhanced VEN to target AML cell lines and primary patient samples while sparing normal hematopoietic cells. Cooperative suppression of OXPHOS was detected in a subset of AML cell lines and primary patient samples. Metabolomics analysis revealed a significant reduction of purine biosynthesis metabolites by ME-344. Further, lometrexol, a purine biosynthesis inhibitor, synergistically enhanced VEN-induced apoptosis in AML cell lines. Interestingly, AML cells with acquired AraC resistance showed significantly increased purine biosynthesis metabolites and sensitivities to ME-344. Furthermore, synergy between ME-344 and VEN was preserved in these AraC-resistant AML cells. In vivo studies revealed significantly prolonged survival upon combination therapy of ME-344 and VEN in NSGS mice bearing parental or AraC-resistant MV4-11 leukemia compared to the vehicle control. This study demonstrates that ME-344 enhances VEN antileukemic activity against preclinical models of AML by suppressing OXPHOS and/or purine biosynthesis.

Smaller Hippocampal Volume Is Associated With Reduced Posttraumatic Stress Symptoms in Children With Cancer and Survivors Following a Brief Novel Martial Arts-Based Intervention.

PURPOSE: Children with cancer and survivors frequently report posttraumatic stress symptoms (PTSS), which are associated with volumetric changes in stress-sensitive brain regions, including the hippocampus. METHODS: We examined the impact of a novel, 4-week martial-arts-based meditative intervention on cancer-related PTSS in 18 pediatric patients and survivors and whether baseline hippocampal volumes correlate with PTSS severity and/or PTSS changes over time. RESULTS: Overall, PTSS did not significantly change from baseline to post-intervention. Smaller hippocampal volume was correlated with more severe re-experiencing PTSS at baseline, and greater reductions in PTSS post-intervention. CONCLUSIONS: Together, hippocampal volume may be a biomarker of PTSS severity and intervention response. Identifying hippocampal volume as a potential biomarker for PTSS severity and intervention response may allow for more informed psychosocial treatments.

Targeting GD2-positive Refractory/Resistant Neuroblastoma and Osteosarcoma with Anti- CD3 x Anti-GD2 Bispecific Antibody Armed T cells.

Background: Since treatment of neuroblastoma (NB) with anti-GD2 monoclonal antibodies provides a survival benefit in children with minimal residual disease and our preclinical study shows that anti-CD3 x anti-GD2 bispecific antibody (GD2Bi) armed T cells (GD2BATs) were highly cytotoxic to GD2+ cell lines, we conducted a phase I/II study in recurrent/refractory patients to establish safety and explore the clinical benefit of GD2BATs. Methods: The 3+3 dose escalation study (NCT02173093) phase I involved 9 evaluable patients with NB (n=5), osteosarcoma (OST) (n=3), and desmoplastic small round cell tumors (DSRCT) (n=1) with twice weekly infusions of GD2BATs at 40, 80, or 160 x 106 GD2BATs/kg/infusion with daily interleukin 2 (300,000 IU/m2) and twice weekly granulocyte-macrophage colony stimulating factor (250 µg/m2). Phase II portion of the trial was conducted in patients with NB at the dose 3 level of 160 x 106 GD2BATs/kg/infusion but failed to enroll the planned number of patients. Results: Nine of 12 patients in the phase I completed therapy. There were no dose limiting toxicities (DLTs). All patients developed mild and manageable cytokine release syndrome (CRS) with grade 2-3 fevers/chills, headaches, and occasional hypotension up to 72 hours after GD2BAT infusions. GD2-antibody associated pain was not significant in this study. The median OS for patients in the Phase I and limited Phase II was 18.0 and 31.2 months, respectively, whereas the combined OS was 21.1 months. There was a complete bone marrow response with overall stable disease in one of the phase I patients with NB. Ten of 12 phase II patients were evaluable for response: 1 had partial response. Three additional patients were deemed to have clinical benefit with prolonged stable disease. More than 50% of evaluable patients showed augmented immune responses to GD2+ targets after GD2BATs as measured by interferon-gamma (IFN-γ) EliSpots, Th1 cytokines, and/or chemokines. Conclusions: Our study demonstrated safety of up to 160 x 106 cells/kg/infusion of GD2BATs. Combined with evidence for the development of post treatment endogenous immune responses, this data supports further investigation of GD2 BATs in larger Phase II clinical trials.

Acquired resistance to venetoclax plus azacitidine in acute myeloid leukemia: In vitro models and mechanisms.

The combination of venetoclax (VEN) and azacitidine (AZA) has become the standard of care for acute myeloid leukemia (AML) patients who are ≥ 75 years or unfit for intensive chemotherapy. Though initially promising, resistance to the combination therapy is an issue and VEN + AZA-relapsed/refractory patients have dismal outcomes. To better understand the mechanisms of resistance, we developed VEN + AZA-resistant AML cell lines, MV4-11/VEN + AZA-R and ML-2/VEN + AZA-R, which show > 300-fold persistent resistance compared to the parental lines. We demonstrate that these cells have unique metabolic profiles, including significantly increased levels of cytidine triphosphate (CTP) and deoxycytidine triphosphate (dCTP), changes in fatty acid and amino acid metabolism and increased utilization and reliance on glycolysis. Furthermore, fatty acid transporter CD36 is increased in the resistant cells compared to the parental cells. Inhibition of glycolysis with 2-Deoxy-D-glucose re-sensitized the resistant cells to VEN + AZA. In addition, the VEN + AZA-R cells have increased levels of the antiapoptotic protein Mcl-1 and decreased levels of the pro-apoptotic protein Bax. Overexpression of Mcl-1 or knockdown of Bax result in resistance to VEN + AZA. Our results provide insight into the molecular mechanisms contributing to VEN + AZA resistance and assist in the development of novel therapeutics to overcome this resistance in AML patients.

Curing childhood cancer the "Natural" Way: Nature as the source of chemotherapy agents.

For many centuries, products of natural origin from plants, marine, microbes and soil micro-organisms have been studied by numerous researchers across the world to yield many of the chemotherapeutic agents we use in this modern era. There has been a tremendous gain in knowledge from various screening and separating techniques which led to the discovery of biologically active small molecules from natural products. Preclinical studies testing the antitumor activities of these agents against tumor cell lines and xenograft animal models were the gateway to the clinical trials in humans leading to the approval of these agents that are in clinical use today. This review summarizes how various chemotherapeutic agents were discovered from products of natural origin, their preclinical development, and their indications in both pediatric and adult oncology. Many of these natural products have contributed to the very high cure rates of both pediatric leukemias and solid tumors.

Enhancing anti-AML activity of venetoclax by isoflavone ME-344 through suppression of OXPHOS and/or purine biosynthesis.

Venetoclax (VEN), in combination with low dose cytarabine (AraC) or a hypomethylating agent, is FDA approved to treat acute myeloid leukemia (AML) in patients who are over the age of 75 or cannot tolerate standard chemotherapy. Despite high response rates to these combination therapies, most patients succumb to the disease due to relapse and/or drug resistance, providing an unmet clinical need for novel therapies to improve AML patient survival. ME-344 is a potent isoflavone with demonstrated inhibitory activity toward oxidative phosphorylation (OXPHOS) and clinical activity in solid tumors. Given that OXPHOS inhibition enhances VEN antileukemic activity against AML, we hypothesized that ME-344 could enhance the anti-AML activity of VEN. Here we report that ME-344 synergized with VEN to target AML cell lines and primary patient samples while sparing normal hematopoietic cells. Cooperative suppression of OXPHOS was detected in a subset of AML cell lines and primary patient samples. Metabolomics analysis revealed a significant reduction of purine biosynthesis metabolites by ME-344. Further, lometrexol, an inhibitor of purine biosynthesis, synergistically enhanced VEN-induced apoptosis in AML cell lines. Interestingly, AML cells with acquired resistance to AraC showed significantly increased purine biosynthesis metabolites and sensitivities to ME-344. Furthermore, synergy between ME-344 and VEN was preserved in these AraC-resistant AML cells. These results translated into significantly prolonged survival upon combination of ME-344 and VEN in NSGS mice bearing parental or AraC-resistant MV4-11 leukemia. This study demonstrates that ME-344 enhances VEN antileukemic activity against preclinical models of AML by suppressing OXPHOS and/or purine biosynthesis.

Co-targeting of HDAC, PI3K, and Bcl-2 results in metabolic and transcriptional reprogramming and decreased mitochondrial function in acute myeloid leukemia.

Despite the recently approved new therapies, the clinical outcomes of acute myeloid leukemia (AML) patients remain disappointing, highlighting the need for novel therapies. Our lab has previously demonstrated the promising outlook for CUDC-907, a dual inhibitor of PI3K and HDAC, in combination with venetoclax (VEN), against AML both in vitro and in vivo at least partially through suppression of c-Myc. In this study, we further elucidated the mechanism of action of the combination in preclinical models of AML. We demonstrated that the combination significantly reduced primary AML cell engraftment in immunocompromised mice. RNA sequencing and metabolomics analyses revealed that the combination reduced the levels for mRNAs of key TCA cycle genes and metabolites in the TCA cycle, respectively. This was accompanied by a reduced oxygen consumption rate (OCR), demonstrating that the combination suppressed oxidative phosphorylation (OXPHOS). Metabolomics analyses revealed that a large number of metabolites upregulated in AraC-resistant AML cells could be downregulated by the combination. CUDC-907 synergized with VEN in inducing apoptosis in the AraC-resistant AML cells. In conclusion, the CUDC-907 and VEN combination induces metabolic and transcriptomic reprograming and suppression of OXPHOS in AML, which provides additional mechanisms underlying the synergy between the two agents.

Inhibition of Mcl-1 Synergistically Enhances the Antileukemic Activity of Gilteritinib and MRX-2843 in Preclinical Models of FLT3-Mutated Acute Myeloid Leukemia.

FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (FLT3-ITD) mutations occur in about 25% of all acute myeloid leukemia (AML) patients and confer a poor prognosis. FLT3 inhibitors have been developed to treat patients with FLT3-mutated AML and have shown promise, though the acquisition of resistance occurs, highlighting the need for combination therapies to prolong the response to FLT3 inhibitors. In this study, we investigated the selective Mcl-1 inhibitor AZD5991 in combination with the FLT3 inhibitors gilteritinib and MRX-2843. The combinations synergistically induce apoptosis in AML cell lines and primary patient samples. The FLT3 inhibitors downregulate c-Myc transcripts through the suppression of the MEK/ERK and JAK2/STAT5 pathways, resulting in the decrease in c-Myc protein. This suppression of c-Myc plays an important role in the antileukemic activity of AZD5991. Interestingly, the suppression of c-Myc enhances AZD5991-inudced cytochrome c release and the subsequent induction of apoptosis. AZD5991 enhances the antileukemic activity of the FLT3 inhibitors gilteritinib and MRX-2843 against FLT3-mutated AML in vitro, warranting further development.

c-Myc plays a critical role in the antileukemic activity of the Mcl-1-selective inhibitor AZD5991 in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive disease with a low 5-year overall survival rate of 29.5%. Thus, more effective therapies are in need to prolong survival of AML patients. Mcl-1 is overexpressed in AML and is associated with poor prognosis, representing a promising therapeutic target. The oncoprotein c-Myc is also overexpressed in AML and is a significant prognostic factor. In addition, Mcl-1 is required for c-Myc induced AML, indicating that c-Myc-driven AML harbors a Mcl-1 dependency and co-targeting of Mcl-1 and c-Myc represents a promising strategy to eradicate AML. In this study, we investigated the role of c-Myc in the antileukemic activity of Mcl-1 selective inhibitor AZD5991 and the antileukemic activity of co-targeting of Mcl-1 and c-Myc in preclinical models of AML. We found that c-Myc protein levels negatively correlated with AZD5991 EC50s in AML cell lines and primary patient samples. AZD5991 combined with inhibition of c-Myc synergistically induced apoptosis in AML cell lines and primary patient samples, and cooperatively targeted leukemia progenitor cells. AML cells with acquired resistance to AZD5991 were resensitized to AZD5991 when c-Myc was inhibited. The combination also showed promising and synergistic antileukemic activity in vitro against AML cell lines with acquired resistance to the main chemotherapeutic drug AraC and primary AML cells derived from a patient at relapse post chemotherapy. The oncoprotein c-Myc represents a potential biomarker of AZD5991 sensitivity and inhibition of c-Myc synergistically enhances the antileukemic activity of AZD5991 against AML.

Meditation reduces brain activity in the default mode network in children with active cancer and survivors.

BACKGROUND: Mounting evidence demonstrates that meditation can lower pain and emotional distress in adults, and more recently, in children. Children may benefit from meditation given its accessibility across a variety of settings (e.g., surgical preparation). Recent neuroimaging studies in adults suggest that meditation techniques are neurobiologically distinct from other forms of emotion regulation, such as distraction, that rely on prefrontal control mechanisms, which are underdeveloped in youth. Rather, meditation techniques may not rely on "top-down" prefrontal control and may therefore be utilized across the lifespan. PROCEDURE: We examined neural activation in children with cancer, a potentially distressing diagnosis. During neuroimaging, children viewed distress-inducing video clips while using martial arts-based meditation (focused attention, mindful acceptance) or non-meditation (distraction) emotion regulation techniques. In a third condition (control), participants passively viewed the video clip. RESULTS: We found that meditation techniques were associated with lower activation in default mode network (DMN) regions, including the medial frontal cortex, precuneus, and posterior cingulate cortex, compared to the control condition. Additionally, we found evidence that meditation techniques may be more effective for modulating DMN activity than distraction. There were no differences in self-reported distress ratings between conditions. CONCLUSION: Together, these findings suggest that martial arts-based meditation modulates negative self-referential processing associated with the DMN, and may have implications for the management of pediatric pain and negative emotion.

"FLipping" the Story: FLT3-Mutated Acute Myeloid Leukemia and the Evolving Role of FLT3 Inhibitors.

The treatment of many types of cancers, including acute myeloid leukemia (AML), has been revolutionized by the development of therapeutics targeted at crucial molecular drivers of oncogenesis. In contrast to broad, relatively indiscriminate conventional chemotherapy, these targeted agents precisely disrupt key pathways within cancer cells. FMS-like tyrosine kinase 3 (FLT3)-encoding a critical regulator of hematopoiesis-is the most frequently mutated gene in patients with AML, and these mutations herald reduced survival and increased relapse in these patients. Approximately 30% of newly diagnosed AML carries an FLT3 mutation; of these, approximately three-quarters are internal tandem duplication (ITD) mutations, and the remainder are tyrosine kinase domain (TKD) mutations. In contrast to its usual, tightly controlled expression, FLT3-ITD mutants allow constitutive, "run-away" activation of a large number of key downstream pathways which promote cellular proliferation and survival. Targeted inhibition of FLT3 is, therefore, a promising therapeutic avenue. In April 2017, midostaurin became both the first FLT3 inhibitor and the first targeted therapy of any kind in AML to be approved by the US FDA. The use of FLT3 inhibitors has continued to grow as clinical trials continue to demonstrate the efficacy of this class of agents, with an expanding number available for use as both experimental standard-of-care usage. This review examines the biology of FLT3 and its downstream pathways, the mechanism of FLT3 inhibition, the development of the FLT3 inhibitors as a class and uses of the agents currently available clinically, and the mechanisms by which resistance to FLT3 inhibition may both develop and be overcome.

The paradox of Myeloid Leukemia associated with Down syndrome.

Children with Down syndrome constitute a distinct genetic population who has a greater risk of developing acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) compared to their non-Down syndrome counterparts. The risk for developing solid tumors is also distinct from the non-Down syndrome population. In the case of myeloid leukemias, the process of leukemogenesis in Trisomy 21 begins in early fetal life where genetic drivers including GATA1 mutations lead to the development of the preleukemic condition, transient abnormal myelopoiesis (TAM). Various other mutations in genes encoding cohesin, epigenetic regulators and RAS pathway can result in subsequent progression to Myeloid Leukemia associated with Down Syndrome (ML-DS). The striking paradoxical feature in the Down syndrome population is that even though there is a higher predisposition to developing AML, they are also very sensitive to chemotherapy agents, particularly cytarabine, thus accounting for the very high cure rates for ML-DS compared to AML in children without Down syndrome. Current clinical trials for ML-DS attempt to balance effective curative therapies while trying to reduce treatment-associated toxicities including infections by de-intensifying chemotherapy doses, if possible. The small proportion of patients with relapsed ML-DS have an extremely poor prognosis and require the development of new therapies.

High-dose AraC is essential for the treatment of ML-DS independent of postinduction MRD: results of the COG AAML1531 trial.

Myeloid leukemia in children with Down syndrome (ML-DS) is associated with young age and somatic GATA1 mutations. Because of high event-free survival (EFS) and hypersensitivity of the leukemic blasts to chemotherapy, the prior Children's Oncology Group protocol ML-DS protocol (AAML0431) reduced overall treatment intensity but lacking risk stratification, retained the high-dose cytarabine course (HD-AraC), which was highly associated with infectious morbidity. Despite high EFS of ML-DS, survival for those who relapse is rare. AAML1531 introduced therapeutic risk stratification based on the previously identified prognostic factor, measurable residual disease (MRD) at the end of the first induction course. Standard risk (SR) patients were identified by negative MRD using flow cytometry (<0.05%) and did not receive the historically administered HD-AraC course. Interim analysis of 114 SR patients revealed a 2-year EFS of 85.6% (95% confidence interval [CI], 75.7-95.5), which was significantly lower than for MRD- patients treated with HD-AraC on AAML0431 (P = .0002). Overall survival at 2 years was 91.0% (95% CI, 83.8-95.0). Twelve SR patients relapsed, mostly within 1 year from study entry and had a 1-year OS of 16.7% (95% CI, 2.7-41.3). Complex karyotypes were more frequent in SR patients who relapsed compared with those who did not (36% vs 9%; P = .0248). MRD by error-corrected sequencing of GATA1 mutations was piloted in 18 SR patients and detectable in 60% who relapsed vs 23% who did not (P = .2682). Patients with SR ML-DS had worse outcomes without HD-AraC after risk classification based on flow cytometric MRD.