Patient-Driven Discovery, Therapeutic Targeting, and Post-Clinical Validation of a Novel AKT1 Fusion–Driven Cancer

Improved knowledge of cancer genomics has revolutionized the way we understand and treat cancer. Recurrent fusion oncoproteins are a hallmark of childhood cancer and have been essential in investigating tumorigenesis and developing therapies (1). The EWS–FLI1 (2) and DNAJB1–PRKACA (3) fusions, for example, are pathognomonic for the specific entities of Ewing sarcoma and fibrolamellar carcinoma, respectively, and assist in precise diagnostics, whereas the BCR–ABL fusion has revolutionized the biological understanding and treatment of specific leukemia subtypes (4). Despite these successes, many pathognomonic fusion oncogenes are not targetable with currently available therapies.

Despite evidence for hyperactivation or mutation of the AKT proteins and their surrounding axis in a multitude of adult-onset and subsets of pediatric malignancies (5–9), only rare oncogenic fusions involving AKT2 (BCAM–AKT2) and AKT3 (RPS6KC1–AKT3) have been described and validated in ovarian cancer and breast cancer, respectively (10, 11). Additionally, a MAGI3–AKT3 fusion was described and initially thought to be recurrent in breast cancer (12), but thereafter could not be validated (13), and was later amended to be noted in one index case only (14). The PI3K/AKT/mTOR pathway regulates metabolism, homeostasis, survival, and proliferation and is now well understood to have a role in the pathogenesis of multiple cancers (15). Despite the central role of AKT1 within this axis, there have thus far been no oncogenic fusions identified involving this gene. AKT1 is an intracellular kinase that is mutated at a low frequency across a broad range of cancers. Of more than 20,000 patient samples that have undergone targeted hybrid-based capture sequencing at our center, the AKT1 gene is mutated in 1.7% across all samples, but in close to 6% of hormone receptor–positive breast cancers and 4% of endometrial cancers (16, 17). Sixty-three percent of these mutations cause a glutamic acid to lysine substitution at amino acid 17 in the PH domain (E17K; refs. 18, 19), which was proven to be a valid therapeutic target in a recent basket trial (20).

In this report, we report the identification of LAMTOR1–AKT1, a novel gene fusion, and describe its patient-driven discovery, therapeutic targeting, and post-clinical validation as a tumorigenic driver and constitutive activator of AKT1.

The patient presented at 3 years of age with abdominal swelling, pain, and elevated CA-125 levels (647 U/mL). Initial pathologic review was inconclusive despite review at multiple institutions with differential diagnoses, including mesothelioma and papillary serous ovarian carcinoma. Over the following 9 years, the patient had multiple surgeries and received numerous lines of systemic therapy, targeted agents including sirolimus, pazopanib, and bevacizumab, immunotherapy with nivolumab, external beam radiation, intraperitoneal chemotherapy, and hyperthermic intraperitoneal chemotherapy. Targeted hybrid capture–based sequencing (21) during the patient's eighth year of treatment revealed mutations in TP53 and the TERT promoter, as well as a rearrangement involving the AKT1 gene that was reported as a variant of unknown significance.

Further examination of two tumor specimens by a second targeted hybrid capture–based sequencing assay (22) and anchored multiplex PCR (23) both confirmed the AKT1 rearrangement and showed that it resulted in an in-frame fusion transcript joining exon 1 of Late endosomal/lysosomal Adaptor, MAPK and mTOR activator 1 (LAMTOR1) to exons 5–14 of AKT1 (Fig. 1A). Structural analysis revealed that this fusion results in N-terminal truncation of AKT1, removing amino acids 1–104, which constitute 96% of the PH domain, a key regulatory domain of AKT1 (Fig. 1A). Disruption of the interaction between the PH and kinase domains has been shown to result in constitutive activation of AKT1, and mutations destabilizing this interaction can be oncogenic (24, 25). Normally, the PH domain maintains AKT1 in an inactive conformation, while also mediating membrane localization in response to PI3K signaling, an essential step for AKT1 activation (26). Notably, the C-terminal LAMTOR1 residues which are part of the fusion were predicted to be myristoylated (27, 28), suggesting that LAMTOR1–AKT1 may be recruited to the membrane in an inappropriately constitutive fashion. Additionally, the described fusion retains the complete kinase domain as well as the key phosphorylation sites T308 and S473 of AKT1 (Fig. 1A), allowing for its activation. The elimination of the PH domain also suggested that the use of allosteric AKT inhibitors, known to require this region to lock AKT1 into an inactive confirmation (29, 30), would have been ineffective. Based on these in silico predictions, the patient was treated with ipatasertib, an ATP-competitive pan-AKT inhibitor, via a compassionate-use mechanism.

Treatment with ipatasertib (300 mg daily) was started when the patient's CA-125 level was 10,065 U/mL in the setting of widespread disease (Fig. 1B-1). The patient tolerated the treatment well and reported a significant decrease in pain and improved energy level within 1 week of beginning ipatasertib. Extent of disease scans obtained at day 26 revealed markedly decreased disease burden and standardized uptake value avidity (Fig. 1B-2). CA-125 levels were also reflective of dramatic improvement in disease burden, decreasing to 7,637, 2,637, and 1,585 U/mL on days 8, 26, and 62 of therapy, respectively (Fig. 1C). Tumor burden was additionally monitored via interrogation of plasma cell-free DNA (cfDNA) using a droplet-digital polymerase chain reaction (ddPCR) assay specific to the patient's novel fusion event (31), and was further reflective of treatment effect (Fig. 1C).

Therapy was well tolerated, with the only noted adverse event being grade 1 diarrhea managed with loperamide and diphenoxylate/atropine. Grade 1 leukopenia with the lowest absolute neutrophil count of 0.8 K/μL at day 8 was noted, but resolved without growth factor support or modification of ipatasertib dosing. Rash and hyperglycemia were not noted.

After approximately 2 months of therapy, the patient's CA-125 level began to rise, and by the fourth month of therapy had reached levels equivalent to those obtained prior to the initiation of ipatasertib (Fig. 1C). Repeat PET/CT imaging revealed recurrence of hypermetabolic disease (Fig. 1B-3). Targeted hybrid capture–based sequencing of a repeat biopsy obtained at this time reidentified the patient's original AKT1 fusion, TP53 and TERT mutations, as well as a new point mutation in STAT3 (c.922A>G), which was present at a low allelic frequency of 0.07% and classified as a variant of unknown significance. Notably, no secondary mutations in AKT1 or LAMTOR1–AKT1 were identified. Due to a lack of targetable mutations potentially responsible for resistance, we hypothesized that tumor progression was due to autocrine/paracrine activation of related pathways, and elected to add temsirolimus to her treatment regimen. Despite the addition of temsirolimus 35 mg/m² given on three occasions, the patient's disease continued to progress and the patient died of disease 5 months after initiation of ipatasertib treatment.

Additional analyses were performed simultaneously with and following the patient's treatment with ipatasertib. Whole-genome sequencing (WGS) and RNA sequencing (RNA-seq) were performed on an excised lymph node with high tumor purity (estimated 75%), obtained approximately 12 weeks after ipatasertib initiation. A total of 7,407 single-nucleotide variants, 641 indels, and 82 rearrangements were detected, for a total mutational burden of 2.77 mutations/Mb (Fig. 2A–D). Notable alterations included deletion of PBRM1 (heterozygous, 10 kb), ROS1, FANCA, and EML4, and missense mutations of unknown significance in CASC5 (c.5237C>T), BCL11B (c.410A>G), STAT3 (c.922A>G), a TP53 splice site (c.97-2AG>TT), and the TERT promoter (c.124C>T). Broad amplifications of chromosome 8 and arm-level losses in 14q, 17p, and 18q were observed. The presence of an unbalanced translocation joining exon 1 of LAMTOR1 and exon 5 of AKT1 was orthogonally validated using RNA-seq analysis (Supplementary Fig. S1A). Four other fusions of unknown significance, including GNL2–EPHA10, STK38–MTCH1, PTGDS–ACTB, and KDM3B–GPR158-AS1, were also identified via RNA-seq (Supplementary Fig. S1B).

Clustering of the corrected variant allele frequencies (VAF) of substitutions identified three predominant clones (Fig. 2E). Significantly, the LAMTOR1–AKT1 fusion was present in the founder clone and was associated with a clonal deletion event on chromosome 14 (Fig. 2E). The TERT promoter mutation was identified as being of potential functional significance, but was subclonal (cluster 2, 48% of cells).

Mutational signature analysis revealed that 70% of the mutations in the founder clone were assigned to signatures S-16 and S-26, which are associated with mismatch-repair deficiency (ref. 32; Supplementary Fig. S1C). The remainder of mutations in the founder clone were assigned to S-P1, a recently described signature detected in a pediatric cohort of patients associated with atypical teratoid rhabdoid tumors (33). These three signatures were absent in subsequent clones. This signature analysis was consistent with clonal evolution in the setting of the patient's long-standing history of high-intensity chemotherapy, in addition to endogenous mutation processes.

To further classify tumor histology, tumor gene expression was compared with samples available in The Cancer Genome Atlas (TCGA) data set (34) and an additional cohort of neuroendocrine tumors (32 tumor types) as previously described (35, 36). Results summarized on two-dimensional t-distributed stochastic neighbor embedding (t-SNE) space (Fig. 2F) and Pearson correlation coefficient heat map (Supplementary Fig. S1D) supported mesothelioma as the closest match of this patient's tumor type, based on the close similarity to malignant pleural mesothelioma samples (TCGA), although overlap with breast and ovarian carcinomas was also noted.

To further characterize this malignancy harboring a LAMTOR1–AKT1 fusion, patient-derived cell (PDCL) and patient-derived xenograft (PDX) models were established from an affected lymph node excised 26 days after the start of ipatasertib treatment (specimen code MSKCAR-65573). Both MSKCAR-65573-PDCL and MSKCAR-65573-PDX models were confirmed to harbor the fusion of interest by immunoblotting analysis (Fig. 3A–C), targeted exome sequencing (Supplementary Fig. S2A), and RT-PCR (Supplementary Fig. S2B). The LAMTOR1–AKT1 fusion appeared as a 48-kDa band recognized by antibodies developed against AKT1 C-terminal epitopes (Ab 28422, OTI 4D6), but not by antibodies recognizing the AKT1 PH domain (CST D9R8K; Fig. 3A–C). Transient expression of LAMTOR1–AKT1 in HEK-293T cells additionally confirmed that the observed band corresponded to the LAMTOR1–AKT1 fusion protein (Supplementary Fig. S2C). Immunofluorescent and IHC analysis of MSKCAR-65573-PDCL (Fig. 3D), patient (Fig. 3E, left), and MSKCAR-65573-PDX (Fig. 3E, right) tissues showed strong positivity for membrane localized AKT1–pS473, consistent with a constitutively active, membrane-bound LAMTOR1–AKT1 fusion protein. Immunofluorescence analysis of AKT1 localization, by the use of an antibody that recognizes only the AKT1 N-terminal domain (Supplementary Fig. S2D), showed that AKT1 is predominantly, if not exclusively, localized in the cytoplasm, supporting our hypothesis that the observed membrane-localized AKT1–pS473 signal was due to the LAMTOR1–AKT1 fusion protein (Supplementary Fig. S2E). Ectopic expression of an eGFP-tagged LAMTOR1–AKT1 fusion protein in HeLa cells formally confirmed plasma membrane localization of LAMTOR1–AKT1 (Fig. 3F). Membrane localization was abolished by mutating the myristoylation and palmitoylation sites on the N-terminus of the LAMTOR1–AKT1 fusion protein (37), demonstrating the need for these post-translational modifications for membrane recruitment (Fig. 3F).

The structural domains preserved in the fusion product predicted susceptibility to ATP-competitive, but not allosteric, inhibitors due to the absence of the AKT1 PH domain recognized by allosteric AKT1 inhibitors (38). To test this hypothesis, MSKCAR-65573-PDCLs were incubated with either ipatasertib or the allosteric AKT inhibitor MK-2206, and cell viability and activity of the AKT1/mTORC1 pathway were evaluated. As expected, ipatasertib inhibited both cell growth (Fig. 3G) and phosphorylation of AKT1 direct target PRAS40 and mTORC1 target RPS6 (Fig. 3H). Increased phosphorylation of AKT1 and LAMTOR1–AKT1 at both the S473 and T308 phosphorlyation sites was noted with ipatasertib treatment, as expected based on previous studies (39). Although the increased phosphorylation in AKT1 was more pronounced on S473, only LAMTOR1–AKT1 T308 phosphorylation was significantly increased by ipatasertib, whereas LAMTOR1–AKT1 S473 remained strongly phosphorylated and unchanged. Notably, the LAMTOR1–AKT1 fusion protein was highly phosphorylated and catalytically active in both the presence and absence of growth factor (B27 supplement), suggesting constitutive activity of the fusion protein (Fig. 3H). MK-2206 only inhibited growth at concentrations >10 μmol/L (Fig. 3G), but did not block LAMTOR1–AKT1 signaling at any concentration tested (Fig. 3H; Supplementary Fig. S2F), indicating that the growth-inhibitory effect observed at high concentrations was due to off-target effects. Ipatasertib and MK-2206 were equally effective in reducing the growth of A204 cells, used as control, confirming that the lack of an effect of MK-2206 in MSKCAR-65573-PDCL was specific (Supplementary Fig. S2G).

As noted above, comparative molecular profiling of the patient's tumor when initially responsive to, and later resistant to, ipatasertib therapy did not identify a genetically encoded mechanism of resistance. In in vitro testing, despite effective inhibition of LAMTOR1–AKT1 activity as shown by reduction of PRAS40 phosphorylation (Fig. 3H), ipatasertib did not completely inhibit tumor growth, and approximately 20% cell viability was still observed at the highest concentration tested (Fig. 3G). Additionally, residual phospho-RPS6 signal was detectable even in the presence of 1 μmol/L ipatasertib (Fig. 3H). We therefore hypothesized that residual mTORC1 activity could be responsible for the observed ipatasertib resistance, and tested various inhibitors acting on the PI3K–AKT1–mTORC1 pathway to identify the potential sources of this activity (Fig. 4A; refs. 15, 40). Among all inhibitors tested, only ipatasertib reduced PRAS40 phosphorylation (Fig. 4B). Inhibition of PI3K (using wortmannin or the more specific BKM120) or PDK1 (GSK2334470), which act upstream of LAMTOR1–AKT1, did not reduce PRAS40 phosphorylation (Fig. 4B; Supplementary Fig. S3A), and only partially reduced RPS6 phosphorylation (Fig. 4B), confirming the constitutive activity of the LAMTOR1–AKT1 fusion protein. As expected, rapamycin, which directly targets the mTORC1 complex downstream of LAMTOR1–AKT1, resulted in more efficient inhibition of mTORC1 activity than ipatasertib or any other inhibitor tested, as indicated by the abolition of RPS6 phosphorylation (Fig. 4B). It has previously been shown that PDK1, through SGK1, regulates mTORC1 in an AKT-independent manner (41), conferring resistance to PI3K inhibitors, and we therefore hypothesized that a similar mechanism could be occurring in this cancer. However, inhibition of SGK1 with GSK650394 (Fig. 4B) or other specific SGK1 inhibitors (Supplementary Fig. S3B) did not reduce RPS6 phosphorylation in these cells. PDK1 inhibition with GSK2334470, however, did reduce RPS6 phosphorylation. This inhibition of mTORC1 activity as a result of inhibition of PDK1, independent of its action on LAMTOR1–AKT1 or SGK1, suggested that PDK1 acts downstream of LAMTOR1–AKT1. This is consistent with phosphorylation of p70-S6K T229 by PDK1, a phosphorylation step that is required for the full activation of p70–S6K and subsequent RPS6 phosphorylation (42). To corroborate these findings, the status of p70-S6K T229 was evaluated in the presence of GSK2334470, with confirmation of reduction in phosphorylation (Supplementary Fig. S3C). PDK1 phosphorylation of p70-S6K T229 has been reported to be independent of PI3K activity (42), in agreement with the observed lack of activity of wortmannin on pRPS6. Moreover, the addition of ipatasertib to the PDK1 inhibitor GSK2334470 did not result in any additive effect on RPS6 phosphorylation (Supplementary Fig. S3D), confirming PDK1 inhibition acted along the same pathway and prevented RPS6 phosphorylation downstream of LAMTOR1–AKT1. Overall, these results indicated that residual mTORC1 activity, as determined by incomplete inhibition of RPS6 phosphorylation, was not due to the activation of LAMTOR1–AKT1 independent pathways, but was the result of incomplete inactivation of LAMTOR1–AKT1 activity by ipatasertib.

These concepts were further interrogated in vivo when mice carrying PDX tumors were administered either ipatasertib, the allosteric inhibitor MK-2206, or the mTORC1 inhibitor temsirolimus. Only ipatasertib and temsirolimus treatments resulted in tumor growth inhibition, whereas MK2206 was ineffective (Fig. 4C). Ipatasertib treatment efficiently inhibited phosphorylation of the AKT1 direct target PRAS40 and partially inhibited the mTORC1 target RPS6 in the treated animals, whereas no changes were observed in the animals that received MK-2206 (Fig. 4D). As was similarly noted above in in vitro testing, temsirolimus achieved more profound inhibition of mTORC1 activity in vivo as shown by marked inhibition of RPS6 phosphorylation, when compared with ipatasertib-treated animals (Fig. 4D). Quantification of cleaved caspase-3 and Ki67 signals after 3 days of treatment showed a statistically significant induction of apoptosis in both ipatasertib- and temsirolimus-treated samples, but only ipatasertib animals experienced a significant reduction in proliferation (Fig. 4E). These results show that inhibition of LAMTOR–AKT1 activity results in both decreased proliferation and increased apoptosis in vivo.

Despite these effects, the responses to both ipatasertib and temsirolimus were transient, and tumor regrowth was noted following approximately 3 weeks of treatment (Supplementary Fig. S3E). At day 40, phosphorylation of both PRAS40 and RPS6 in ipatasertib-treated animals remained suppressed, but not abolished, confirming ongoing ipatasertib inhibitory activity (Supplementary Fig. S3F). To further identify possible mechanisms of resistance to ATP-competitive AKT inhibition, we utilized a reverse phase protein array assay to compare ipatasertib-treated animals at 3 and 40 days of treatment and directly evaluated MEK/ERK pathway activity. There was no clear upregulation of ERK activity or alternative pathways (Supplementary Fig. S3G; Supplementary Tables S1–S5), confirming that tumor regrowth could have resulted from the residual mTORC1 activity observed in ipatasertib-treated animals. Overall, in vitro and in vivo observations suggested that combination therapy with ATP-competitive AKT inhibition as well as mTORC1 inhibition would have been necessary to provide a complete and sustained inhibition of the AKT1/mTORC1 signaling axis and tumor growth.

To formally test the hypothesis, we first evaluated the effect of wortmannin, GSK2334470, and rapamycin on cell growth as single agents. GSK2334470 and rapamycin showed inhibition of cell growth as single agents, whereas wortmannin did not significantly reduce cell growth (Supplementary Fig. S4A). These findings are concordant with our biochemical analysis showing that wortmannin, contrary to GSK2334470 or rapamycin, did not reduce RPS6 phosphorylation. When ipatasertib administration, in combination with either GSK233470 or rapamycin, was analyzed using the Chou–Talalay method (43), an additive effect was noted for both (Supplementary Fig. S4B). These observations further support our model that incomplete inactivation of the mTOR pathway downstream of LAMTOR1–AKT1 is responsible for the observed resistance.

We further proceeded to evaluate if the addition of mTORC1 inhibition could overcome resistance to ipatasertib in vivo. MSKCAR-65573-PDX models were treated with ipatasertib or temsirolimus as single agents as well as in combination (Fig. 4F). Tumors treated with either agent alone were again noted to regrow after approximately 3 weeks of treatment, whereas combination therapy inhibited tumor growth as long as animals were dosed (approximately 4 months). To confirm that tumor growth in ipatasertib-treated animals was due to inefficient inhibition of mTORC1 activity, temsirolimus was administered to mice that had been treated with ipatasertib only, and an immediate inhibition of tumor growth was observed (Fig. 4G). Additionally, tumors treated with temsirolimus similarly responded to the addition of ipatasertib. These results, taken in combination with the lack of secondary mutations in AKT1 or other candidate genes in both the patient and the MSKCAR-65573-PDX samples sequenced during periods of ipatasertib resistance (data not shown), as well as lack of upregulation of ERK or other pathways in ipatasertib-resistant MSKCAR-65573-PDX tumors, corroborate residual mTORC1 activity as a mediator of resistance to ATP-competitive AKT inhibition in this LAMTOR1–AKT1 malignancy.

This report describes a novel, activating AKT1 fusion, as well as the first use of the AKT inhibitor ipatasertib in a pediatric patient. This experience highlights the promise of real-time incorporation of molecular profiling results into clinical care, underscores the necessity of increased access to early-phase agents for pediatric patients, and reveals a novel, targetable oncogenic fusion within a critical signaling pathway.

The activating LAMTOR1–AKT1 fusion described here results in the removal of nearly the entire PH domain and is therefore hypothesized to disrupt the inhibitory interaction between the PH and kinase domain in a manner similar to that described for E17K mutations (18, 24, 44). Loss of the PH domain would also result in the inability of the fusion to be recruited to the plasma membrane, where AKT1 is normally activated by PDK1- and mTORC2-mediated phosphorylation. However, the presence of myristoylation and palmitoylation sites in the LAMTOR1 fusion moiety leads to LAMTOR1–AKT1 localization to the plasma membrane, thus functionally rescuing the loss of the PH domain. A similar mechanism of activation has been described for the BCAM–AKT2 fusion oncoprotein (10), strongly suggesting that membrane localization of AKT fusion proteins is a requirement for their oncogenic activity.

Stratification of adult-onset cancers has benefited from ever more rapid and cost-effective molecular profiling, but analyses of pediatric cancers have not yielded similar successes for children and young adults. This disparity has been traditionally attributed to the relatively undisturbed pediatric cancer genome in the setting of a paucity of applicable and available compounds. This experience adds to several prominent examples including the use of assembly-based whole-genome DNA sequencing to discover previously undefined rearrangements in rhabdoid tumors (45), and the finding of genomic rearrangements in the telomerase reverse transcriptase gene (TERT; ref. 46) in high-risk neuroblastoma, which highlight the need for broad and comprehensive approaches to molecular profiling, particularly in pediatric cancers. Analysis of WGS revealed that the overall mutational burden (2.77 subs/Mb) in this patient's tumor was higher than typical for a majority of pediatric cancers (32, 47), most likely due to significant pretreatment with known genotoxic agents including platinum compounds. In spite of this relatively high mutational burden, there was a rapid and dramatic clinical response to ipatasertib treatment, supporting this novel fusion as the primary oncogenic driver in this cancer.

It is most notable that the use of ipatasertib in this patient was driven by rapid, in silico analyses with the ensuing hypothesis that the identified LAMTOR1–AKT1 fusion could be an oncogenic driver. The compassionate use of ipatasertib in this setting had not been validated in either the preclinical or clinical setting and, furthermore, had not been administered to a pediatric patient. Instead, it was this patient's experience itself, followed by what can be referred to as “post-clinical validation,” that now corroborates these initial hypotheses. Under such circumstances, it would not have been practical or beneficial to the patient to proceed in the traditional, and reverse, fashion, allowing time for preclinical confirmation before administering the drug. With the deluge of ever more quickly available molecular profiling, we suspect such opportunities will become routine, and a paradigm to proceed from sequencing or other similar personalized data to individualized clinical trial, followed by or simultaneous with post-clinical validation, will be accepted even in pediatric patients.

The coming together of multiple clinical, research, and regulatory platforms and sources of expertise facilitated the discovery, therapeutic targeting, and characterization of the first described oncogenic AKT1 gene fusion. The first-ever pediatric use of a well-tolerated, orally bioavailable, ATP-competitive AKT kinase inhibitor, ipatasertib, validated this molecular event as an oncogenic driver, and was successful in inducing a dramatic clinical response. However, as is common with single-agent targeted inhibition, resistance was noted and precluded durable response. Our experience is complementary to the results of an ipatasertib phase I clinical trial in adult-onset cancers, in which the most responsive patients carried alterations in the PI3K pathway, but achieved only stable disease at best (20). Our post-clinical analyses provide insights into possible mechanisms of resistance to single-agent ipatasertib therapy, suggesting that in these LAMTOR1–AKT1 fusion–driven models, ipatasertib treatment as a single agent was not sufficient to completely inhibit mTORC1 activity, and that this residual activity precluded more durable response. These observations suggest that combination ATP-competitive AKT and mTORC1 inhibition is rational, may be successful in overcoming resistance to single-agent AKT inhibition, and can be considered for future clinical trials focused on malignancies with analogous AKT activating mutations. We note that although we administered the combination of ipatasertib and temsirolimus in the described patient following progression on ipatasertib alone, these agents were given in combination very briefly during a period of profound illness secondary to multiorgan failure in the setting of widespread disease progression, and her experience during this period must therefore be interpreted with caution. Additionally, the patient did receive sirolimus (in combination with bevacizumab and irinotecan, and later in combination with pazopanib) prior to her exposure to ipatasertib, and some transient stability of disease was noted on both regimens, followed by progression of disease. Further work is warranted to achieve a more comprehensive understanding of the biological implications of the described fusion and the prevalence and role of this or other AKT1 fusions in human cancers.

Written informed consent and verbal assent were obtained from the patient's guardian and the patient, respectively, for treatment with ipatasertib. All studies were conducted in accordance with recognized ethical guidelines including the Belmont Report and the U.S. Common Rule, and all studies were approved by an institutional review board.

pBABE–puro retroviral vector was purchased from Addgene. Ipatasertib, MK-2206, temsirolimus, SGK233470, wortmannin, rapamycin, and SGK650394 were purchased from Selleckchem. SGK1 inhibitors compound 14g and mp-1 were provided by Dr. Marc Nazare (Leibniz-Forschungsinstitut für Molekulare Pharmakologie). Unless specified, all in vitro treatments were performed using 1 μmol/L concentration for 3 hours.

The PDX model (code: MSKCAR-65573, specimen 0002a) was developed by transplanting a tumor sample from the patient's lymph node into the flank of NOD (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ) SCID gamma (NSG) mice to generate the passage 0 (P0) generation. Upon reaching a tumor width of ∼1 cm, the PDX P0 tumors were collected and expanded into a larger cohort. Animals were dosed with single agents ipatasertib 100 mg/kg/day (oral gavage), or temsirolimus 20 mg/kg/day (intraperitoneal injection), or MK-2206 100 mg/kg/day(oral gavage). When combination treatment was administered, doses were reduced to ipatasertib 75 mg/kg/day and temsirolimus 10 mg/kg 3 days per week to avoid excessive toxicity. All experiments were performed in accordance with institutional guidelines and under an approved protocol from The Memorial Sloan Kettering Cancer Center Institutional Animal Care and Use Committee (protocol number 16-08-011).

HEK-293T and HeLa cells were obtained from ATCC and were maintained in DMEM, 10% FBS, penicillin/streptomycin, and fungizone with a doubling time of ∼72 hours. MSKCAR-65573-CL cells were generated from the MSKCAR-65573-PDX p0 model by harvesting tumors, mincing into 2–4 mm pieces, and dissociating using the Tumor Dissociation Kit (MACS, Miltenyi Biotec) on a MACS Octo Dissociator with Heaters (Miltenyi Biotec) at 37°C for 60 minutes under continuous rotation. The cell line MSKCAR-65573-CL was established in neurobasal media + B27 supplement. All cells were tested for Mycoplasma as previously described (48) approximately once every 4 months (last performed October 2018).

The coding sequences of AKT1, LAMTOR1–AKT1 fusion construct, and mutant LAMTOR1–AKT1 (G2A or C3S, C4S) were synthesized by GenScript in vector pUC57. Coding sequences were cloned into the pcDNA3.1-C-eGFP expression vector between the BamHI and KpnI restriction sites in frame with N-terminal eGFP. Transfection of 293T or HeLa cells was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer's guidelines.

Cells (2,500 cells/well) were seeded in 96-well plates and after 24 hours were incubated with single drugs (ipatsertib, MK-2206, GSK1904529A, wortmannin, GSK2334470, rapamycin, KU-0063794, or GSK650394) for 72 hours. The viability of the cells was determined using CellTiter-Glo Luminescent Cell Viability Assay (Promega). IC₅₀ concentrations were determined using GraphPad Prism Software.

Targeted hybridization capture DNA sequencing, anchored multiplex RNA-seq, WGS, RNA-seq, and t-SNE are described in detail in Supplementary Materials and Methods.

These investigations were carried out as has been previously described (31).

F.S. Dela Cruz is a consultant/advisory board member for Shasqi Inc., Y-mAbs Therapeutics, Syros Pharmaceuticals, and Eisai. L.M. Smyth reports receiving a commercial research grant from AstraZeneca, reports receiving other commercial research support from AstraZeneca, PUMA Biotechnology, and Roche Genentech, has received honoraria from the speakers bureaus of AstraZeneca and Pfizer, is a consultant/advisory board member for Roche Genentech, and has received other remuneration from PUMA Biotechnology, Pfizer, and Roche Genentech. R. Darnell is Founding Director and CEO of New York Genome Center and is a consultant/advisory board member for Atreca and MITRE. A. Califano is founder, equity holder, consultant, and director of DarwinHealth Inc., a company that has licensed some of the algorithms used in this article from Columbia University. Columbia University is also an equity holder in DarwinHealth Inc. E. Papaemmanuil reports receiving a commercial research grant from Celgene and has received honoraria from the speakers bureau of TRTH mentoring. D.M. Hyman reports receiving commercial research grants from Loxo Oncology, PUMA Biotechnology, AstraZeneca, and Bayer Pharmaceuticals, and is a consultant/advisory board member for Atar Biotherapeutics, Chugai Pharma, CytomX Therapeutics, Boehringer Ingelheim, AstraZeneca, Pfizer, Bayer Pharmaceuticals, and Genentech/Roche. No potential conflicts of interest were disclosed by the other authors.

Conception and design: E.K. Slotkin, D. Diolaiti, N.N. Shukla, F.S. Dela Cruz, L.M. Smyth L. Spraggon, A. Califano, A.L. Kung, D.M. Hyman, S.S. Roberts

Development of methodology: D. Diolaiti, N.N. Shukla, F.S. Dela Cruz, D. You, A. Califano, A.L. Kung, S.S. Roberts

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): E.K. Slotkin, D. Diolaiti, N.N. Shukla, F.S. Dela Cruz, J.J. Clark, D. You, P. Ma, S. Pachhal, G. Ibanez Sanchez, R. Benayed, A.A. Jungbluth, L.M. Smyth, I. Gushterova, L. Spraggon, R. Darnell, M. Ladanyi, A.L. Kung, D.M. Hyman, S.S. Roberts

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): E.K. Slotkin, D. Diolaiti, N.N. Shukla, F.S. Dela Cruz, G. Gundem, V.D. Yellapantula, M.F. Levine, D. You, P. Ma, S. Pachhal, G. Ibanez Sanchez, A. Mauguen, I. Gushterova, H. Ding, R. Darnell, A. Califano, E. Papaemmanuil, A.L. Kung

Writing, review, and/or revision of the manuscript: E.K. Slotkin, D. Diolaiti, N.N. Shukla, F.S. Dela Cruz, D. You, L.M. Smyth, A. Mauguen,A. Califano, M. Ladanyi, E. Papaemmanuil, A.L. Kung, D.M. Hyman, S.S. Roberts

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D. You, D.M. Hyman

Study supervision: E.K. Slotkin, D. Diolaiti, L. Spraggon, M. Ladanyi,A.L. Kung, S.S. Roberts

Other (performed IHC for this study): A.A. Jungbluth

The authors would like to thank ArcherDX, Inc. and Array BioPharma for assistance in characterizing the novel LAMTOR1–AKT1 fusion, Roche/Genentech for supplying ipatasertib, Dr. Elisa de Stanchina and the MSKCC Antitumor Assessment Core Facility for assistance in performing in vivo testing, Dr. Marc Nazare for providing SGK1 inhibitors mp-1 and cmp-14, Jessie Hillsberg for her caring treatment of the patient, and Drs. Todd Heaton and Michael P. LaQuaglia for their support in obtaining biopsies. Funding for this work was generously provided by the Sohn Conference Foundation, The PaulieStrong Foundation, Cycle for Survival (Team Kate in memory of Kate Hennemuth Reap), the Olayan Precision Pediatrics Program, and the NCI grant P30 CA008748. This work was supported by the NCI Cancer Target Discovery and Development Program (U01 CA217858), an NCI Outstanding Investigator Award (R35 CA197745), and NIH Shared Instrumentation Grants (S10 OD012351 and S10 OD021764), all to A. Califano.

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