Project Confirm: Accelerated Drug Approvals for Chronic Myeloid Leukemia.

The FDA has an accelerated approval program for drugs that have been identified as promising treatments for serious conditions when the available data suggest that the benefits outweigh the foreseeable risks. All of the currently available treatment options for chronic myeloid leukemia (CML) initially went through the accelerated approval program. Here, a group of academic CML experts, patient panelists, and members from the FDA convened to discuss the utility of the accelerated approval program as it pertains to CML, and the utility of this program in future drug development in this disease. The results of that discussion are summarized here.

OTS167 blocks FLT3 translation and synergizes with FLT3 inhibitors in FLT3 mutant acute myeloid leukemia.

Internal tandem duplication (-ITD) mutations of Fms-like tyrosine kinase 3 (FLT3) provide growth and pro-survival signals in the context of established driver mutations in FLT3 mutant acute myeloid leukemia (AML). Maternal embryonic leucine zipper kinase (MELK) is an aberrantly expressed gene identified as a target in AML. The MELK inhibitor OTS167 induces cell death in AML including cells with FLT3 mutations, yet the role of MELK and mechanisms of OTS167 function are not understood. OTS167 alone or in combination with tyrosine kinase inhibitors (TKIs) were used to investigate the effect of OTS167 on FLT3 signaling and expression in human FLT3 mutant AML cell lines and primary cells. We describe a mechanism whereby OTS167 blocks FLT3 expression by blocking FLT3 translation and inhibiting phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and eukaryotic translation initiation factor 4B (eIF4B). OTS167 in combination with TKIs results in synergistic induction of FLT3 mutant cell death in FLT3 mutant cell lines and prolonged survival in a FLT3 mutant AML xenograft mouse model. Our findings suggest signaling through MELK is necessary for the translation and expression of FLT3-ITD, and blocking MELK with OTS167 represents a viable therapeutic strategy for patients with FLT3 mutant AML.

New Approaches to Treating Challenging Subtypes of ALL in AYA Patients.

PURPOSE OF REVIEW: The treatment of acute lymphoblastic leukemia (ALL) in adolescent and young adult (AYA) patients has markedly improved with the adoption of pediatric-inspired protocols. However, there remain several subtypes of ALL that represent significant therapeutic challenges. Here, we review the current evidence guiding treatment of Philadelphia chromosome-positive (Ph+), Philadelphia chromosome-like (Ph-L), and early T-precursor (ETP) ALL in the AYA population. RECENT FINDINGS: Clinical trials in Ph + ALL have demonstrated the superior efficacy of second- and third-generation tyrosine kinase inhibitors (TKIs) to induce and maintain remission. Current efforts now focus on determining the durability of these remissions and which patients will benefit from transplant. For Ph-like and ETP ALL, recent studies are investigating the addition of novel agents to standard treatment. The treatment of Ph + ALL has significantly improved with the addition of potent TKIs. However, the treatment of Ph-like and ETP ALL remains a challenge. At this time, the judicious use of allogenic transplant is the only current approach to modify this increased risk.

Efficacy and toxicity of reduced vs. standard dose pegylated asparaginase in adults with Philadelphia chromosome-negative acute lymphoblastic leukemia.

Incorporation of asparaginase (ASNase) and pegylated asparaginase (PEG-ASP) into pediatric-inspired regimens for adults with acute lymphoblastic leukemia (ALL) has led to improved treatment outcomes albeit with increased toxicities. This study compared the efficacy and safety of the Children's Oncology Group standard PEG-ASP (SD) dosing (>1000, median 2500 IU/m2/dose) in adult Philadelphia chromosome-negative ALL patients receiving multiagent chemotherapy vs reduced dose PEG-ASP (RED) (≤1000, median 500 IU/m2/dose) during induction. 51 patients were included, 26 in RED and 25 in SD (median age 49 vs 37 years, p = .027). Median day 7 ASNase activity level for RED was 0.16 IU/mL. All 11 patients who received PEG-ASP 1000 IU/m2 and 9/11 patients who received 500 IU/m2 achieved an ASNase level ≥0.1 IU/mL. Patients receiving RED experienced fewer total grade 3/4 toxicities during induction compared to SD (p = .02) while still attaining therapeutic ASNase levels. RED permits safer ASNase use in adults with ALL and should be tested in a larger cohort prospectively.

Effective breast cancer combination therapy targeting BACH1 and mitochondrial metabolism.

Mitochondrial metabolism is an attractive target for cancer therapy1,2. Reprogramming metabolic pathways could improve the ability of metabolic inhibitors to suppress cancers with limited treatment options, such as triple-negative breast cancer (TNBC)1,3. Here we show that BTB and CNC homology1 (BACH1)4, a haem-binding transcription factor that is increased in expression in tumours from patients with TNBC, targets mitochondrial metabolism. BACH1 decreases glucose utilization in the tricarboxylic acid cycle and negatively regulates transcription of electron transport chain (ETC) genes. BACH1 depletion by shRNA or degradation by hemin sensitizes cells to ETC inhibitors such as metformin5,6, suppressing growth of both cell line and patient-derived tumour xenografts. Expression of a haem-resistant BACH1 mutant in cells that express a short hairpin RNA for BACH1 rescues the BACH1 phenotype and restores metformin resistance in hemin-treated cells and tumours7. Finally, BACH1 gene expression inversely correlates with ETC gene expression in tumours from patients with breast cancer and in other tumour types, which highlights the clinical relevance of our findings. This study demonstrates that mitochondrial metabolism can be exploited by targeting BACH1 to sensitize breast cancer and potentially other tumour tissues to mitochondrial inhibitors.

Inotuzumab: from preclinical development to success in B-cell acute lymphoblastic leukemia.

Inotuzumab ozogamicin (InO) is a recently US Food and Drug Administration-approved antibody-drug conjugate for the treatment of relapsed/refractory B-cell acute lymphoblastic leukemia (ALL). InO consists of a CD22-targeting immunoglobulin G4 humanized monoclonal antibody conjugated to calicheamicin. Although initially developed for the treatment of non-Hodgkin lymphoma (NHL) because of activity in preclinical models and high response rates in indolent lymphomas, a phase 3 trial was negative and further development focused on CD22+ ALL. Although results in NHL were disappointing, parallel testing in early-phase trials of CD22+ ALL demonstrated feasibility and efficacy. Subsequently, the randomized phase 3 Study Of Inotuzumab Ozogamicin Versus Investigator's Choice Of Chemotherapy In Patients With Relapsed Or Refractory Acute Lymphoblastic Leukemia trial showed that InO was superior to standard of care regimens with a significantly improved complete remission (CR) rate in patients with relapsed/refractory disease (80.7% vs 29.4%, P < .001). Patients achieving CR with InO also had a significantly higher rate of undetectable minimal residual disease compared with chemotherapy (78.4% vs 28.1%, P < .001). InO-specific side effects, including veno-occlusive disease, have been an ongoing area of concern, and consensus guidelines for minimizing toxicities are now available. Ongoing trials are investigating the combination of InO with other agents in the relapse setting and the addition of InO to frontline therapy. This review details the preclinical and clinical development of InO, focusing on how best to use it and future directions for further development.

Rap1 and its effector RIAM are required for lymphocyte trafficking.

Regulation of integrins is critical for lymphocyte adhesion to endothelium and trafficking through secondary lymphoid organs. Inside-out signaling to integrins is mediated by the small GTPase Rap1. Two effectors of Rap1 regulate integrins, RapL and Rap1 interacting adaptor molecule (RIAM). Using mice conditionally deficient in both Rap1a and Rap1b and mice null for RIAM, we show that the Rap1/RIAM module is not required for T- or B-cell development but is essential for efficient adhesion to intercellular adhesion molecule (ICAM) 1 and vascular cell adhesion molecule (VCAM) 1 and for proper trafficking of lymphocytes to secondary lymphoid organs. Interestingly, in RIAM-deficient mice, whereas peripheral lymph nodes (pLNs) were depleted of both B and T cells and recirculating B cells were diminished in the bone barrow (BM), the spleen was hypercellular, albeit with a relative deficiency of marginal zone B cells. The abnormality in lymphocyte trafficking was accompanied by defective humoral immunity to T-cell-dependent antigens. Platelet function was intact in RIAM-deficient animals. These in vivo results confirm a role for RIAM in the regulation of some, but not all, leukocyte integrins and suggest that RIAM-regulated integrin activation is required for trafficking of lymphocytes from blood into pLNs and BM, where relatively high shear forces exist in high endothelial venules and sinusoids, respectively.

Metabolic labeling of Ras with tritiated palmitate to monitor palmitoylation and depalmitoylation.

Metabolic labeling with tritiated palmitate is a direct method for monitoring posttranslational modification of Ras proteins with this fatty acid. Advances in intensifying screens have allowed for the easy visualization of tritium without the need for extended exposure times. While more energetic radioisotopes are easier to visualize, the lack of commercial source and need for shielding make them more difficult to work with. Since radiolabeled palmitate is directly incorporated into Ras, its loss can be monitored by traditional pulse-chase experiments that cannot be accomplished with the method of acyl-exchange chemistry. As such, tritiated palmitate remains a readily accessible and direct method for monitoring the palmitoylation status of Ras proteins under a multitude of conditions.

Rap1-interacting adapter molecule (RIAM) associates with the plasma membrane via a proximity detector.

Adaptive immunity depends on lymphocyte adhesion that is mediated by the integrin lymphocyte functional antigen 1 (LFA-1). The small guanosine triphosphatase Rap1 regulates LFA-1 adhesiveness through one of its effectors, Rap1-interacting adapter molecule (RIAM). We show that RIAM was recruited to the lymphocyte plasma membrane (PM) through its Ras association (RA) and pleckstrin homology (PH) domains, both of which were required for lymphocyte adhesion. The N terminus of RIAM inhibited membrane translocation. In vitro, the RA domain bound both Rap1 and H-Ras with equal but relatively low affinity, whereas in vivo only Rap1 was required for PM association. The PH domain bound phosphoinositol 4,5-bisphosphate (PI(4,5)P(2)) and was responsible for the spatial distribution of RIAM only at the PM of activated T cells. We determined the crystal structure of the RA and PH domains and found that, despite an intervening linker of 50 aa, the two domains were integrated into a single structural unit, which was critical for proper localization to the PM. Thus, the RA-PH domains of RIAM function as a proximity detector for activated Rap1 and PI(4,5)P(2).

Phospholipase D1 regulates lymphocyte adhesion via upregulation of Rap1 at the plasma membrane.

Rap1 is a small GTPase that modulates adhesion of T cells by regulating inside-out signaling through LFA-1. The bulk of Rap1 is expressed in a GDP-bound state on intracellular vesicles. Exocytosis of these vesicles delivers Rap1 to the plasma membrane, where it becomes activated. We report here that phospholipase D1 (PLD1) is expressed on the same vesicular compartment in T cells as Rap1 and is translocated to the plasma membrane along with Rap1. Moreover, PLD activity is required for both translocation and activation of Rap1. Increased T-cell adhesion in response to stimulation of the antigen receptor depended on PLD1. C3G, a Rap1 guanine nucleotide exchange factor located in the cytosol of resting cells, translocated to the plasma membranes of stimulated T cells. Our data support a model whereby PLD1 regulates Rap1 activity by controlling exocytosis of a stored, vesicular pool of Rap1 that can be activated by C3G upon delivery to the plasma membrane.