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

Identification of a LAMTOR1–AKT1 Fusion

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.

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Figure 1.

A novel AKT1 fusion–driven cancer. A, Schematic diagram of the genomic DNA, mRNA, and protein structures of the LAMTOR1–AKT1 t(11,14)(q.13.4, q32.33) fusion. Top, fusion breakpoints within intron 1 of LAMTOR1 (red) and exon 5 of AKT1 (blue); middle, the resulting fusion mRNA structure; bottom, the protein structure, including the near-total removal of AKT1 PH domain, the retention of the entire kinase domain, and both phosphorylation sites (T308 and S473). B, Representative CT images of the chest and whole-body projections from ¹⁸fluorodeoxyglucose (FDG)-PET scans are shown. Plot 1 depicts tumor burden at the start of treatment with the AKT inhibitor ipatasertib; plot 2 depicts repeat imaging following 4 weeks of treatment with ipatasertib; plot 3 depicts repeat imaging following 10 weeks of therapy. C, Dual axis graph comparing levels of the patient's established tumor marker, CA-125, and the number of LAMTOR1–AKT1 fusion–positive droplets per mL of plasma via a cfDNA assay over time.

Antitumor Efficacy of Ipatasertib

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.

Pan-Genomic Tumor Characterization and Classification

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).

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Figure 2.

Pan-genomic analysis of a novel AKT1 fusion–driven pediatric cancer. A, Circos plot summarizing WGS analysis with the LAMTOR1–AKT1 fusion marked by a red arrow; the two innermost tracks depict the integer copy-number changes for the major (brown) and minor (dark pink) allele; the outermost track shows the intermutation distance for substitutions each plotted according to the type of nucleotide change; the middle track shows the genomic positions of the small insertion (green) and deletion (red) along the genome; rearrangements are plotted as arcs inside the circos; labeled genes show aberrations in genes present in the Cancer Genes Census. B, Mutation signature analysis using deconstructSigs. C, Summary of the insertion and deletion data. D, Summary of rearrangement data. E, Distribution of corrected VAF identifying the LAMTOR1–AKT1 fusion as a founder clonal event.F, Scatter plot showing the t-SNE 2-D projection of 2,400 samples, including at least 75 samples (indicated in the figure) for each of the 32 tissue types represented; the described patient's tissue is indicated by a black triangle; tumor-type abbreviations: ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast carcinoma; CESC, cervical carcinoma; COAD, colon adenocarcinoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck carcinoma; KICH, kidney chromophobe; KIRC, clear cell carcinoma of the kidney; KIRP, renal papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, low-grade glioma; LIHC, hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MENI, meningioma; MESO, mesothelioma; NET, gastrointestinal neuroendocrine tumor; OV, ovarian carcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectal adenocarcinoma; SARC, sarcoma; SKCM, cutaneous melanoma; STAD, gastric adenocarcinoma; TGCT, testicular germ cell tumor; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma; UVM, uveal melanoma.

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.

Generation and Validation of Patient-Derived Cells and Xenograft Models

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).

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Figure 3.

Generation and validation of PDCL and PDX models and confirmation of LAMTOR1–AKT1 membrane localization. A, Schematic localization of the epitopes recognized by the indicated anti-AKT1 antibodies (CST-D9R8K, Ab-28422, and OTI-4D6) which were used to confirm the presence of the LAMTOR1–AKT1 fusion in PDCL and PDX models, B, Immunoblotting analysis confirming the expression of the LAMTOR1–AKT1 fusion protein in the PDCL (MSKCAR-65573-PDCL); cells derived from a previously described undifferentiated carcinoma (CUCAR-11182) expressing AKT1 were used as control. C, Immunoblotting analysis confirming the expression of the LAMTOR1–AKT1 fusion protein in the PDX tissue (MSKCAR-65573). D, Immunofluorescence of AKT1–pS473 in PDCL. E, Plot 1 depicts a high-powered view of IHC analysis of AKT1–pS473 (brown) in the patient's sample confirming marked membrane localization; plots 2 and 3 depict low- and high-powered views, respectively, of immunofluorescent analysis of AKT1–pS473 (green) and its plasma membrane localization in the PDX (MSKCAR-65573-PDCL) model; plot 4 depicts IgG control. F, Plots 1–5 depict representative images of HeLa cells transfected with plasmids encoding the indicated proteins fused to eGFP (N-terminus; blue, Hoechst 33342; red, LysoTraker DND-99; green, GFP or eGFP labeled protein); LysoTraker DND-99 red staining was included as control to evaluate lysosomal localization; the sequence depicted in the box represents the first 14 amino acids of the LAMTOR1–AKT1 fusion including the myristoylation and palmitoylation sites; plots 4 and 5 depict the lack of membrane localization following mutation of the noted myristoylation and palmitoylation sites. G, Evaluation of the antiproliferative effect of ipatasertib and MK-2206 on the PDX model (MSKCAR-65573-PDCL) in vitro. H, Immunoblotting analysis of the indicated proteins in the PDX model (MSKCAR-65573-PDCL) upon treatment with ipatasertib or MK-2206 in the presence and absence of serum growth factor B27.

LAMTOR1–AKT1 Is Susceptible to ATP-Competitive AKT Inhibition

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).

LAMTOR1–AKT1 Resistance to Ipatasertib Is Mediated through Residual mTORC1 Activity

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.

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Figure 4.

Dual inhibition of LAMTOR1–AKT1 and mTORC1 is necessary to inhibit tumor growth. A, Schematic representation of the PI3K–AKT–mTORC1 axis and the target of each inhibitor. SGKi, SGK inhibitor. B, Immunoblotting analysis of selected proteins in the presence of the indicated inhibitors. C, Waterfall plot showing PDX; MSKCAR-65573) tumor size changes after 15 days of treatment (best response) with either ipatasertib, MK-2206, or temsirolimus; response was classified as progressive disease (>50% tumor volume increase), stable disease (between 50% increase and 50% reduction), or partial response (>50% tumor volume reduction), and each column represents a single animal. D, Immunoblotting analysis of indicated proteins in tumors after 3 days of treatment with ipatasertib (Ipa), MK2206 (MK), temsirolimus (Tem), or vehicle (Veh). E, IHC quantification of apoptosis [cleavedcaspase-3 signal (Cl.Cas3)] and proliferation (Ki67 signal) in tumor tissues treated for 3 days with the indicated drugs; mean and standard deviation of tumor tissue obtained from three independent mice per group are shown, and one-way ANOVA analysis is used; *, P < 0.05; **, P < 0.01; ***, P < 0.001. F, Growth curve plot of tumor response to continuous vehicle, ipatasertib, temsirolimus, or ipatasertib + temsirolimus treatments; #, a decrease in tumor volume at day 109 due to the censoring of 2 mice having ulcerated tumors. G, Growth curve plot of tumor volume changes after interruption of treatment in the combination arm or addition of ipatasertib or temsirolimus to the reciprocal single-agent treatments; tumor volumes were normalized to tumor volumes recorded at the time treatment was modified (day 109); *, P < 0.05; **, P < 0.01; ***, P < 0.001; the Fisher exact test was used to evaluate the statistical significance of the difference between groups in E; area under the curve analysis was used for F.

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.