Kaufmann, Scott H.

Abstract The mammalian target of rapamycin (mTOR), a kinase that regulates proliferation and apoptosis, has been extensively evaluated as a therapeutic target in hematologic malignancies. Rapamycin analogues, which partially inhibit mTOR complex 1 (mTORC1), showed limited anti-tumor activity due to feedback mechanisms involving mTORC2 and incomplete inhibition of mTORC1. Thus, attention has turned to agents targeting both mTOR complexes by binding the mTOR kinase domain. The purpose of this study was to delineate the mechanisms of mTOR dual inhibitor induced apoptosis in human neoplastic lymphoid cells in vitro. MTS assays and propidium iodide staining followed by flow cytometry for subdiploid populations demonstrated that OSI-027 and MLN0128 inhibited cell proliferation and induced apoptosis in a wide range of lymphoid cell lines including Jurkat, Nalm-6, Molt-4, and SeAX. Raptor and Rictor knockdown in Jurkat and Nalm-6 increased cell death, suggesting both mTOR complexes play a role in apoptosis. 4EBP1 phosphorylation was inhibited by mTOR dual inhibitors, but not by rapamycin. Expression of 4EBP1 T37A/T46A and 4EBP1 T37A/T46A/S65A/T70A, which mimic dephosphorylated 4EBP1, increased Puma mRNA and protein levels as well as apoptosis. Moreover, 4EBP1 knockdown abrogated mTOR dual inhibitor induced Puma upregulation and cell death, further supporting the role of 4EBP1 dephosphorylation in mTORC1 dependent apoptosis. In accord with the known dependence of c-Myc translation on the eIF4E/eIF4G complex, we also observed c-Myc downregulation after treatment with OSI-027, MLN0128 and 4EGI-1, but not rapamycin. Puma induction mirrored c-Myc downregulation under a variety of conditions, including expression of nonphosphorylatable 4EBP1 in parental Jurkat cells or wt 4EBP1 in 4EBP1 deficient cells. Furthermore, c-Myc knockdown induced Puma mRNA and protein as well as increased apoptosis. Collectively, these results support a model in which mTORC1 inhibition, acting through 4EBP1, induces Puma upregulation and apoptosis through c-Myc downregulation. In order to assess the parallel mTORC2-dependent Bim-mediated apoptotic mechanism, we utilized reporter assays and RNAseq experiments. OSI-027-induced Bim promoter activity decreased markedly when the nucleotides -29 to -18 were removed, suggesting that this response element is critical for OSI-027-induced promoter activation. In silico analysis identified eight transcription factors, including SP1, Egr-1, and Myb, that potentially bind this 12-bp region. In RNAseq experiments, we detected a 9-fold increase in Egr-1. Egr-1 upregulation was confirmed by qRT-PCR and immunoblotting after dual inhibitors treatment or Rictor knockdown. Moreover, dominant negative Egr-1 or Egr-1 knockdown diminished dual inhibitor-induced Bim promoter activation and Bim upregulation. Chromatin immunoprecipitation assays demonstrated that OSI-027 enhances binding of Egr-1 to a region of the Bim promoter including bp -29 to -18, further confirming that Egr-1 functions as a direct transcriptional activator for Bim upon mTOR dual inhibitor treatment. NFκB is a known transcription factor for Egr-1 and we observed increased p65 in the nucleus and increased NFκB transcriptional activity after dual inhibitor treatment. Overexpression of S32A/S36A IκB impaired the ability of dual inhibitors to induce NFκB transcriptional activation, Egr-1 mRNA and protein, Bim promoter activation and Bim mRNA and protein upregulation. Collectively, these results suggest that mTORC2 inhibition induces Bim upregulation and apoptosis through NFκB and Egr-1 transactivation. When fresh clinical ALL isolates were exposed to OSI-027 or MLN0128 ex vivo, inhibition of 4EBP1 phosphorylation along with upregulation of Egr-1 and Bim and/or c-Myc downregulation accompanied by Puma induction occurred, indicating that the pathways identified in ALL cell lines can also potentially be engaged in clinical ALLs. These observations not only provide new insight into the survival roles of mTOR in lymphoid malignancies, but also identify alterations that potentially modulate the action of mTOR dual inhibitors. Disclosures No relevant conflicts of interest to declare.

MLN4924, a novel chemotherapeutic agent designed to inhibit the Nedd8 Activating Enzyme (NAE), has shown promise in early clinical testing in relapsed and refractory Acute Myeloid Leukemia (AML). NAE inhibition prevents transfer of the Nedd8 protein to Cullin Ring Ligases (CRLs), leading to their inactivation and subsequent accumulation of the protein substrates they normally ubiquitinate and target for degradation by the proteasome. In malignant cells treated with MLN4924, various CRL substrates accumulate and induce a cytotoxic effect. Importantly, the substrates responsible for these effects vary among different malignancies depending on basal gene expression patterns. In order to shed light on the mechanism of MLN4924 anti-leukemic effects and identify potential mechanisms of resistance, the effects of MLN4924 on AML cell lines and primary AML samples were examined in vitro and in vivo. These studies identified c-Myc as the critical substrate governing the cytotoxic effect. In particular, we observed c-Myc transactivation of the PMAIP1 gene, leading to expression of pro-apoptotic protein Noxa, which functions to neutralize Mcl-1, activate Bak and Bax to allow release of cytochrome c from the outer mitochondrial membrane, and induce apoptosis. Importantly, MLN4924-induced Noxa upregulation was prevented by c-Myc shRNA; and MLN4924-induced killing was markedly diminished by Noxa siRNA. In further experiments, a synergistic cytotoxic effect was seen when MLN4924 was applied to AML cell lines in combination with agents that inhibit anti-apoptotic proteins other than Mcl-1, such as BH3 mimetics ABT-263 (inhibits Bcl-2, Bcl-xL, and Bcl-2) and ABT-199 (inhibits Bcl-2). Because expression of anti-apoptotic proteins within leukemia samples and between different AMLs is variable, and Mcl-1 upregulation often contributes to drug resistance and relapse in AML, this combination may be advantageous in several clinical settings. To follow up on the effects we observed with MLN4924 alone and in combination with BH3 mimetics, we have designed a multi-parameter flow cytometry based assay to assess response to these agents among hematopoietic stem cells (HSCs) and progenitor populations in primary leukemia bone marrow isolates treated ex vivo. Pilot studies conducted in mononuclear cell fractions containing normal HSCs and hematopoietic progenitors isolated from umbilical cord blood did not undergo apoptosis in response to MLN4924. Ongoing studies aim to specifically assess whether MLN4924 alone or in combination with other anti-leukemic agents induce apoptosis or alter expression of CD47 (an anti-phagocytic protein frequently upregulated in AML) in HSCs and hematopoietic progenitor cell populations within primary AML bone marrow isolates. As our understanding of how HSCs and hematopoietic progenitors contribute to AML initiation and progression advances, the need to target various populations will emerge and may vary depending on disease stage. It will be critical to ascertain whether HSCs and other populations are sensitive or resistant to agents such as MLN4924 and others in order to formulate and apply efficient targeted therapies. Disclosures No relevant conflicts of interest to declare.

Abstract Background: Myelofibrosis with myeloid metaplasia (MMM) is a progressive and fatal myeloproliferative disorder with limited therapeutic options. R115777 is a non-peptidomimetic enzyme-specific inhibitor of farnesyl protein transferase (FT) which we recently reported has in vitro activity against aberrant myeloid progenitors from MMM patients (Leukemia2003;17:849). This observation, coupled with the clinical activity demonstrated in high risk acute myeloid leukemia (Blood2001;97:3361) and chronic myeloid disorders (Blood2003;101:1692) led us to perform a Phase II trial in patients (pts) with MMM. Methods: Eligible pts had histologically confirmed MMM and were symptomatic, defined by anemia (hemoglobin<10g/dL or transfusion dependent) or palpable hepatosplenomegaly. Pts received 300 mg of R115777 orally twice daily for the first 21 days of a 28 day cycle. Dose escalations (up to 1200mg/day) and reductions (down to 100mg/day) were allowed based on activity and toxicity. The primary endpoint was response as defined by improvement in either anemia or organomegaly. Results: 34 pts (median age 65 yrs, range 55–80; 71% male) were enrolled in the trial. All pts displayed symptomatic disease at enrollment, with 10 who were erythrocyte transfusion dependent; and 19 (56%) had a Lille MMM prognostic score ≥1; no pts were significantly thrombocytopenic at entry by enrollment criteria. Median pre-study spleen and liver sizes were 12 cm (range: 0–35) and 2 cm (range: 0–15) below the left and right costal margin, respectively. Median time to discontinuation of therapy was 5.5 months (95% CI:2.9–6.4). Reasons for early termination were progression (7), adverse reactions (5), co-morbidities (2), refusal of further treatment (2), and death on study (1). One pt died of grade 5 pneumonitis two weeks after study enrollment, which was felt to be unrelated to study treatment. Significant toxicities (≥ grade 3) were seen for myelosuppression (n=13), neuropathy (n=2), fatigue (n=1), rash (n=1) and hyponatremia (n=1). Overall, R115777 resulted in little improvement in anemia, with the exception of one anemia-based partial response. However, the myelosuppressive aspects of the agent may have masked improvements in cytopenias. In contrast, R115777 did result in clinically relevant decreases in organomegaly in 11 pts (33%) (splenomegaly in 3 pts, hepatomegaly in 5 pts, and both in an additional 3 pts.) Across all pts who had some reduction in splenomegaly, the median maximal reduction was 42.5% (of palpable component; range: 8–100%). Likewise, all patients with baseline hepatomegaly had resolution of this problem at some point during therapy. Four pts remain on the agent (from 14 to 22 months) for continued and sustained improvement in organomegaly. Responses observed did not significantly correlate with reductions in bone marrow fibrosis, osteosclerosis, neoangiogenesis, or resolution of baseline karyotypic abnormalities. Conclusions: R115777 has demonstrated preliminary activity against MMM associated organomegaly. Whether this latter benefit is independent of R115777 induced myelosuppression is unclear. The myelosuppression observed was generally mild and without clinical consequence, although it was difficult to differentiate between myelosuppression versus disease progression. Pts continue to be followed to evaluate durability of observed responses, long term effects on anemia and intramedullary manifestations of the disease.